The role of transcriptional activation in p53 function is highly controversial. To define this role in vivo, we generated a Trp53 knock-in construct encoding a protein carrying mutations of two residues that are crucial for transactivation (L25Q,W26S). Here we show that these mutations have selective effects on the biological functions of p53. Although its ability to activate various p53 target genes is largely compromised, the p53^QS protein retains the ability to transactivate the gene Bax. The ability of the p53^QS mutant protein to elicit a DNA damage–induced G1 cell cycle–arrest response is also partially impaired. p53^QS has selective defects in its ability to induce apoptosis: it is completely unable to activate apoptosis in response to DNA damage, is partially unable to do so when subjected to serum deprivation and retains substantial apoptotic activity upon exposure to hypoxia. These findings suggest that p53 acts through distinct, stimulus-specific pathways to induce apoptosis. The importance of the biological activity of p53^QSin vivo is underscored by our finding that expression of p53^QS, which cannot bind mdm2, induces embryonic lethality. Taken together, these results suggest that p53 has different mechanisms of action depending on specific contextual cues, which may help to clarify the function of p53 in preventing cancer.