Research on cell senescence and immortalization of murine embryonic fibroblasts (MEFs) has revealed important clues about genetic control of senescence in humans. To investigate senescence and genetic alterations in the p53 pathway that lead to senescence bypass in culture, we compared the behavior of MEFs from wild-type mice with MEFs from Hupki mice, which harbor a humanized p53 gene. We found that humanizing the p53 gene in mice preserved major features of the MEF senescence/immortalization process. In both genotypes, a significant proportion of spontaneously arising cell lines had sustained either a p53 point mutation or p19/ARF biallelic deletion. The p53 mutations selected for during Hupki MEF immortalization have been found in human tumors and are classified in the yeast transactivation assay as transcriptionally defunct, suggesting that disabling this component of p53 activity is crucial in senescence bypass. Surprisingly, in spontaneously immortalized cell lines from both wild-type and Hupki MEFs, the predominant type of p53 mutation was a G to C transversion, rather than the G to T substitutions expected from the raised oxygen levels characteristic of standard culture conditions. Over half of the cell lines did not reveal evidence of p53 mutation or loss of p19/ARF and retained a robust wild-type p53 response to DNA damage, supporting the inference from senescence bypass screens that alternative genetic routes to immortalization occur.