p21 is a CDK Inhibitor regulated by the tumor suppressor p53 and is hypothesized to mediate G1 arrest. p53 has been suggested to derive anti-oncogenic properties from this relationship. To test these notions, we created mice lacking p21. They develop normally and (unlike p53 mice) have not developed spontaneous malignancies during 7 months of observation. Nonetheless, p21 embryonic fibroblasts are significantly deficient in their ability to arrest in G1 In response to DNA damage and nucleotide pool perturbation. p21 cells also exhibit a significant growth alteration in vitro, achieving a saturation density as high as that observed In p53-/- cells. In contrast, other aspects of p53 function, such as thymocytic apoptosis and the mitotic spindle checkpoint, appear normal. These results establish the role of p21 in the G1 checkpoint, but suggest that the antiapoptotic and the anti-oncogenic effects of p53 are more complex. © 1995.