Abstract Pathways mediated by p53 and Rb are frequently altered in aggressive human cancers, including prostate carcinoma. To test directly the roles of p53 and Rb in prostate carcinogenesis, we have conditionally inactivated these genes in the prostate epithelium of the mouse. Inactivation of either p53 or Rb leads to prostatic intraepithelial neoplasia developing from the luminal epithelium by 600 days of age. In contrast, inactivation of both genes results in rapidly developing (median survival, 226 days) carcinomas showing both luminal epithelial and neuroendocrine differentiation. The resulting neoplasms are highly metastatic, resistant to androgen depletion from the early stage of development, and marked with multiple gene expression signatures commonly found in human prostate carcinomas. Interestingly, gains at 4qC3 and 4qD2.2 and loss at 14qA2-qD2 have been consistently found by comparative genomic hybridization. These loci contain such human cancer–related genes as Nfib, L-myc, and Nkx3.1, respectively. Our studies show a critical role for p53 and Rb deficiency in prostate carcinogenesis and identify likely secondary genetic alterations. The new genetically defined model should be particularly valuable for providing new molecular insights into the pathogenesis of human prostate cancer. (Cancer Res 2006; 66(16): 7889-98)