The tumor suppressor p53 is the most frequently mutated gene in human cancer. In vivo models have been generated using knock-in alleles in which missense mutations are introduced that mimic the kinds of mutations found in human cancers, or that abolish specific p53 functions. Critically, these studies examine the in vivo and physiological functions of p53. Studies indicate that p53 missense mutations in the DNA-binding domain identical with those inherited in the Li-Fraumeni syndrome, have distinct properties. Studies in mice with mutants that separate cell-cycle arrest and apoptosis functions of p53 show delayed onset of tumor development, suggesting that both p53 functions are crucial for suppressing tumors. Mice with mutations at post-translational modification sites exhibit subtle effects on p53 activity and tumor development, indicating a fine-tuning mechanism of p53 activity in vivo. Importantly, each mutant mouse has a distinct phenotype, suggesting diverse and exquisite mechanisms of p53 regulation in different environments, different tissues and different genetic backgrounds. The generation of these mutant p53 knock-in mice has laid the groundwork for future studies to elucidate the in vivo physiological function of mutant p53 and to examine cooperating effects in combination with other alterations.