The mammalian G1-S phase transition is controlled by the opposing forces of cyclin-dependent kinases (CDK) and the retinoblastoma protein (pRB). Here, we present evidence for systems-level control of cell cycle arrest by pRB-E2F and p27-CDK regulation. By introducing a point mutant allele of pRB that is defective for E2F repression (Rb1G) into a p27KIP1 null background (Cdkn1b-/-), both E2F transcriptional repression and CDK regulation are compromised. These double-mutant Rb1G/G; Cdkn1b-/- mice are viable and phenocopy Rb1+/- mice in developing pituitary adenocarcinomas, even though neither single mutant strain is cancer prone. Combined loss of pRB-E2F transcriptional regulation and p27KIP1 leads to defective proliferative control in response to various types of DNA damage. In addition, Rb1G/G; Cdkn1b-/- fibroblasts immortalize faster in culture and more frequently than either single mutant genotype. Importantly, the synthetic DNA damage arrest defect caused by Rb1G/G; Cdkn1b-/- mutations is evident in the developing intermediate pituitary lobe where tumors ultimately arise. Our work identifies a unique relationship between pRB-E2F and p27-CDK control and offers in vivo evidence that pRB is capable of cell cycle control through E2F-independent effects.