The recent discovery of checkpoint kinases has suggested the conservation of checkpoint mechanisms between yeast and mammals. In yeast, the protein kinase Chk1 is thought to mediate signaling associated with the DNA damage checkpoint of the cell cycle. However, the function of Chk1 in mammals has remained unknown. Targeted disruption of Chk1 in mice showed thatChk1 ^−/− embryos exhibit gross morphologic abnormalities in nuclei as early as the blastocyst stage. In culture, Chk1 ^−/− blastocysts showed a severe defect in outgrowth of the inner cell mass and died of apoptosis. DNA replication block and DNA damage failed to arrest the cell cycle before initiation of mitosis inChk1 ^−/− embryos. These results may indicate that Chk1 is indispensable for cell proliferation and survival through maintaining the G₂ checkpoint in mammals.