Abstract: A method for inducing differentiation of a pluripotent stem cell into an ectodermal cell, that includes coating a cell culture substrate having regions (A) and (B) below with Matrigel or the like, and seeding the pluripotent stem cell, (A) an island-shaped region having cell adhesiveness or the like, and an adjacent region (B) having no cell adhesiveness or the like; forming a pluripotent stem cell aggregate adhering onto the cell culture substrate by adherently culturing the seeded pluripotent stem cell in a feeder-free manner in an undifferentiated state maintenance medium; forming an embryoid body by culturing the formed pluripotent stem cell aggregate in a medium containing a differentiation-inducing factor in a state of adhering onto the cell culture substrate; and forming an ectodermal cell aggregate by culturing the formed embryoid body in a medium containing a differentiation-inducing factor in a state of adhering onto the cell culture substrate.