Heterozygosity of the retinoblastoma gene Rb1 elicits tumorigenesis in susceptible tissues following spontaneous loss of the remaining functional allele. Inactivation of previously studied retinoblastoma protein (pRb) targets partially inhibited tumorigenesis in Rb1(+/-) mice. In this report AECC members Zhu and Locker report that inactivation of pRb target Skp2 completely prevents spontaneous tumorigenesis in Rb1(+/-) mice. Targeted Rb1 deletion in melanotrophs ablated the entire pituitary intermediate lobe when Skp2 was inactivated. Skp2 inactivation did not inhibit aberrant proliferation of Rb1-deleted melanotrophs but did induce their apoptotic death. Eliminating p27 phosphorylation on T187 in p27T187A knock-in mice reproduced the effects of Skp2 knockout, identifying p27 ubiquitination by SCF(Skp2) ubiquitin ligase as the underlying mechanism for Skp2's essential tumorigenic role in this setting. RB1-deficient human retinoblastoma cells also underwent apoptosis after Skp2 knockdown; and ectopic expression of p27, especially the p27T187A mutant, induced apoptosis. These findings indicate that Skp2 becomes an essential survival gene when susceptible cells incur Rb1 deficiency.