Pesellino was raised by his grandfather, the painter Giuliano il Pesello, and worked as his assistant until Giuliano's death. He then became associated with Filippo Lippi (Lippi, Fra Filippo). In 1453 he went into partnership with Piero di Lorenzo di Pratese, and during this period he began, for the Church of the Trinità at Pistoia, the altarpiece now in the National Gallery, London. It was left unfinished at his death. The “Trinity” altarpiece is the only firmly attributed painting by Pesellino.

Pesellino is famous for his cassone pictures, which were intended for the decoration of marriage chests and in which he illustrated old legends or tales in tapestry-like designs. Several of these are in the Gardner Museum, Boston. A number of works by Pesellino may be seen in the National Gallery of Art, Washington, D.C.; the Metropolitan Museum of Art, New York City; and the Museum of Art of Toledo, Ohio.