Willem van Aelst was born in Delft to a prominent family. He was a pupil from his uncle, the still-life painter Evert van Aelst. On 9 November 1643 he became a member of the Guild of Saint Luke in Delft.
Between 1645 and 1649 he was active in France. In 1649 Van Aelst travelled to Florence, where he served as court painter to Ferdinando II de' Medici, grand duke of Tuscany and the future Cosimo III. At the same time, two other Dutch painters, Matthias Withoos and Otto Marseus van Schrieck, were also employed by the Grand Duke.
In 1656 he returned to the Netherlands together with Van Schrieck, to settle permanently in Amsterdam, although Houbraken mentions that he settled in Delft. He became one of the most prominent still-life painters of his generation. He probably died in 1683 as several works dated 1682 survived, and a work dated 1683 has been reported. Rachel Ruysch was one of his pupils.