The name Masaccio is a humorous version of Maso (short for Tommaso), meaning "big", "fat", "clumsy" or "messy" Tom. The name may have been created to distinguish him from his principal collaborator, also called Maso, who came to be known as Masolino ("little/delicate Tom").

Despite his brief career, he had a profound influence on other artists. He was one of the first to use Linear perspective in his painting, employing techniques such as vanishing point in art for the first time. He also moved away from the International Gothic style and elaborate ornamentation of artists like Gentile da Fabriano to a more naturalistic mode that employed perspective and chiaroscuro for greater realism.

Masaccio was born to Giovanni di Simone Cassai and Jacopa di Martinozzo in Castel San Giovanni di Altura, now San Giovanni Valdarno (today part of the province of Arezzo, Tuscany). His father was a notary and his mother the daughter of an innkeeper of Barberino di Mugello, a town a few miles south of Florence. His family name, Cassai, comes from the trade of his paternal grandfather Simone and granduncle Lorenzo, who were carpenters - cabinet makers ("casse", hence "cassai"). His father died in 1406, when Tommaso was only five; in that year a brother was born, called Giovanni (1406–1486) after the dead father. He also was to become a painter, with the nickname of lo Scheggia meaning "the splinter." In 1412 Monna Jacopa married an elderly apothecary, Tedesco di maestro Feo, who already had several daughters, one of whom grew up to marry the only other documented painter from Castel San Giovanni, Mariotto di Cristofano (1393–1457).

There is no evidence for Masaccio's artistic education. Renaissance painters traditionally began an apprenticeship with an established master at about the age of 12; Masaccio would likely have had to move to Florence to receive his training, but he was not documented in the city until he joined the painters guild (the Arte de' Medici e Speziali) as an independent master on January 7, 1422, signing as "Masus S. Johannis Simonis pictor populi S. Nicholae de Florentia."

The first works attributed to Masaccio are the San Giovenale Triptych (1422) and the Virgin and Child with Saint Anne (Sant'Anna Metterza)(c. 1424) at the Uffizi.

The San Giovenale altarpiece was only discovered in 1961 in the church of San Giovenale at Cascia di Reggello, which is very close to Masaccio's hometown. It represents the Virgin and Child with angels in the central panel, Sts. Bartholomew and Blaise on the left panel, and Sts. Juvenal (i.e. San Giovenale) and Anthony Abbot in the right panel. The painting has lost much of its original framing, and its surface is badly abraded.. Nevertheless, Masaccio's concern to suggest three-dimensionality through volumetric figures and foreshortened forms (a revival of Giotto's approach, rather than a continuation of contemporary trends) is already apparent.

The second work was perhaps Masaccio's first collaboration with the older and already-renowned artist, Masolino da Panicale (1383/4-c. 1436). The circumstances of the 2 artists' collaboration are unclear; since Masolino was considerably older, it seems likely that he brought Masaccio under his wing, but the division of hands in the Virgin and Child with Saint Anne is so marked - Masolino is believed to have painted the figure of St. Anne and the angels that hold the cloth of honor behind her, while Masaccio painted the more important Virgin and Child on their throne - that it is hard to see the older artist as the controlling figure in this commission. Masolino's figures are delicate, graceful and somewhat flat, while Masaccio's are solid and hefty.

In Florence, Masaccio could study the works of Giotto and become friends with Brunelleschi and Donatello. According to Vasari, at their prompting in 1423 Masaccio travelled to Rome with Masolino: from that point he was freed of all Gothic and Byzantine influence, as may be seen in his altarpiece for the Carmelite Church in Pisa. The traces of influences from ancient Roman and Greek art that are present in some of Masaccio's works presumably originated from this trip: they should also have been present in a lost Sagra, (today known through some drawings, including one by Michelangelo), a fresco commissioned for the consecration ceremony of the church of Santa Maria del Carmine in Florence (April 19, 1422). It was destroyed when the church's cloister was rebuilt at the end of the 16th century.

In 1424 the "duo preciso e noto" ("well and known duo") of Masaccio and Masolino was commissioned by the powerful and rich Felice Brancacci to execute a cycle of frescoes for the Brancacci Chapel in the church of Santa Maria del Carmine in Florence. Painting began around 1425 with the two artists probably working simultaneously. For reasons that are unclear they left the chapel unfinished, and it was completed by Filippino Lippi in the 1480s. The iconography of the fresco decoration is somewhat unusual; while the majority of the frescoes represent the life of St. Peter, 2 scenes, on either side of the threshold of the chapel space, depict the temptation and expulsion of Adam and Eve. As a whole the frescoes represent human sin and its redemption through the actions of Peter, the first pope. The style of Masaccio's scenes shows the influence of Giotto especially. Figures are large, heavy, and solid; emotions are expressed through faces and gestures; and there is a strong impression of naturalism throughout the paintings. Unlike Giotto, however, Masaccio uses linear and atmospheric perspective, directional light, and chiaroscuro, which is the representation of form through light and color without outlines. As a result his frescoes are even more convincingly lifelike than those of his trecento predecessor.

The Expulsion from the Garden of Eden, depicts a distressed Adam and Eve, chased from the garden by a threatening angel. Adam covers his face to express his shame, while Eve's shame requires her to cover her body. The fresco had a huge influence on Michelangelo. Another major work is The Tribute Money in which Jesus and the Apostles are depicted as neo-classical archetypes. Scholars have often noted that the shadows of the figures all fall away from the chapel window, as if the figures are lit by it; this is an added stroke of verisimilitude and further tribute to Masaccio's innovative genius. In the Resurrection of the Son of Theophilus he painted a pavement in perspective, framed by large buildings to obtain a three-dimensional space in which the figures are placed proportionate to their surroundings. In this he was a pioneer in applying the newly discovered rules of perspective.