Chk2 is a protein kinase that is activated in response to DNA damage and may regulate cell cycle arrest. We generated Chk2-deficient mouse cells by gene targeting. Chk2(-/-) embryonic stem cells failed to maintain gamma-irradiation-induced arrest in the Ci phase of the cell cycle. Chk2(-/-) thymocytes were resistant to DNA damage-induced apoptosis. Chk2(-/-) cells were defective for p53 stabilization and for induction of p53-dependent transcripts such as p21 in response to gamma irradiation. Reintroduction of the Chk2 gene restored p53-dependent transcription in response to gamma irradiation. Chk2 directly phosphorylated p53 on serine 20, which is known to interfere with Mdm2 binding. This provides a mechanism for increased stability of p53 by prevention of ubiquitination in response to DNA damage.