Gillis Mostaert was born in Hulst. According to the early artist biographer Karel van Mander Gillis Mostaert was the son of a common painter, the twin brother of the painter Frans Mostaert and the grandson of the painter Jan Mostaert. While his brother Frans became a pupil of the landscape painter Herri met de Bles, Gillis studied landscape painting with Jan Mandijn. Van Mander recounts that both brothers joined the Antwerp Guild of Saint Luke in 1555. However, since Frans received a registered pupil in 1553 it is more likely that Frans had become a master in the Guild earlier.
He married Margareta Baes in 1563 and the couple had six children. He was well-respected by his fellow painters. The painters Pieter Balten and Crispin van den Broeck were godfathers to his children born in 1571 and 1588. Inventories of art collections from his time show that his work received patronage of well-off private clients since they contained many expensive cabinet paintings of Mostaert.