Posttranslational modification such as phosphorylation of p53 plays important roles in activating p53 responses to various cellular and genotoxic stresses. Cell line studies have shown that phosphorylation of Ser46 is correlated with the activation of p53 apoptotic activity. To address the physiological roles of Ser46 phosphorylation, we employed homologous recombination and LoxP/Cre-mediated deletion to introduce Ser46 to Ala missense mutation into the human p53 knock-in (HUPKI) allele in mice (p53hki(S46A)). p53 stabilization in response to various types of DNA damage is modestly reduced in p53hki(S46A) embryonic stem (ES) cells, mouse embryonic fibroblasts (MEFs) and thymocytes. In addition, p53-dependent apoptosis is partially impaired in p53hki(S46A) thymocytes and E1A/Ras-expressing mouse embryonic fibroblasts (MEFs) after DNA damage. Consistent with this finding, transcription of p53 target apoptotic genes is preferentially affected by S46A mutation after DNA damage. p53hki(S46A) MEFs proliferate and reach senescence normally but can be spontaneously immortalized more easily than wild type MEFs. In addition, p53hki(S46A) MEFs more readily escapes from Ras-induced senescence. Therefore, Ser46 phosphorylation activates p53-dependent apoptosis induced by DNA damage and cellular senescence induced by oncogenic stress.