Differentiation of a large number of embryonic stem cells or artificial pluripotent stem cells such as induced pluripotent stem cells can be readily induced by the following method. A substrate surface having a cell non-adhesive region on the periphery of a cell adhesive region is used, and(1) the cells are allowed to adhere to the cell adhesive region of the substrate surface in a differentiation-inducing medium(2) the cells are subsequently allowed to grow on the cell adhesive surface to reach confluence(3) the culture is further continued to three-dimensionally stratify the cells from the boundary with the cell non-arlhesive region surrounding the cell adhesive region and(4) a plurality of the stratified portions formed in the cell adhesive region is finally bound to one another to produce a cell agglomerate spreading over the entire cell adhesive region with a certain thickness.
