An object of the invention is to provide a new method which enables efficient induction and manufacturing of definitive endoderm cells and insulin-producing cells from pluripotent stem cells.The present invention provides a method for manufacturing definitive endoderm cells from pluripotent stem cells, which comprises performing a first culture of pluripotent stem cells in a differentiation-inducing medium under conditions where insulin acts, and then performing a second culture in a differentiation-inducing medium under conditions where insulin does not act.本發明係以提供可有效率地從多潛能幹細胞誘導、製造胚胎內胚層細胞或產生胰島素的細胞的新穎的手法為目的。本發明提供一種從多潛能幹細胞製造胚胎內胚層細胞的方法，該方法包含將多潛能幹細胞於胰島素會作用的條件下的分化誘導培養基中進行第1培養，接著，於胰島素不會作用的條件下的分化誘導培養基中進行第2培養。
