The mammalian Chk2 kinase is thought to mediate ATM‐dependent signaling in response to DNA damage. The physiological role of mammalian Chk2 has now been investigated by the generation of Chk2‐deficient mice. Although Chk2−/− mice appeared normal, they were resistant to ionizing radiation (IR) as a result of the preservation of splenic lymphocytes. Thymocytes and neurons of the developing brain were also resistant to IR‐induced apoptosis. The IR‐induced G 1/S cell cycle checkpoint, but not the G 2/M or S phase checkpoints, was impaired in embryonic fibroblasts derived from Chk2−/− mice. IR‐induced stabilization of p53 in Chk2−/− cells was 50–70% of that in wild‐type cells. Caffeine further reduced p53 accumulation, suggesting the existence of an ATM/ATR‐dependent but Chk2‐independent pathway for p53 stabilization. In spite of p53 protein stabilization and phosphorylation of Ser23, p53‐dependent transcriptional induction of target genes, such as p21 and Noxa, was not observed in Chk2−/− cells. Our results show that Chk2 plays a critical role in p53 function in response to IR by regulating its transcriptional activity as well as its stability.