According to Karel van Mander, Mostaert's father was from Hulst, near Antwerp. According to the RKD, he was a painter, but he sent his sons to his own father Jan Mostaert in Haarlem. Gillis had a twin brother, Frans Mostaert, who also became a painter and who looked so much like him that their own parents could not tell them apart. After their early training, Gillis studied with Jan Mandijn and Frans with Herri met de Bles. The Mostaert brothers both joined the Antwerp Guild of Saint Luke in 1554. Perhaps they returned to Antwerp with their grandfather who later died there.

Gillis painted a variety of subjects including religious, battle scenes, and depictions of village life. He married Margareta Baes in 1563 and they went on to hav...