Genome endoreduplication during mammalian development is a rare event for which the mechanism is unknown. It first appears when fibroblast growth factor 4 (FGF4) deprivation induces differentiation of trophoblast stem (TS) cells into the nonproliferating trophoblast giant (TG) cells required for embryo implantation. Here we show that RO3306 inhibition of cyclin-dependent protein kinase 1 (CDK1), the enzyme required to enter mitosis, induced differentiation of TS cells into TG cells. In contrast, RO3306 induced abortive endoreduplication and apoptosis in embryonic stem cells, revealing that inactivation of CDK1 triggers endoreduplication only in cells programmed to differentiate into polyploid cells. Similarly, FGF4 deprivation resulted in CDK1 inhibition by overexpressing two CDK-specific inhibitors, p57/KIP2 and p21/CIP1. TS cell mutants revealed that p57 was required to trigger endoreduplication by inhibiting CDK1, while p21 suppressed expression of the checkpoint protein kinase CHK1, thereby preventing induction of apoptosis. Furthermore, Cdk2(-/-) TS cells revealed that CDK2 is required for endoreduplication when CDK1 is inhibited. Expression of p57 in TG cells was restricted to G-phase nuclei to allow CDK activation of S phase. Thus, endoreduplication in TS cells is triggered by p57 inhibition of CDK1 with concomitant suppression of the DNA damage response by p21.