A large number of human cancers display alterations in theInk4a/cyclin D/Cdk4 genetic pathway, suggesting that activation of Cdk4 plays an important role in oncogenesis. Here we report thatCdk4-null mouse embryonic fibroblasts are resistant to transformation in response to Ras activation with dominant-negative (DN) p53 expression or in the Ink4a/Arf-null background, judged by foci formation, anchorage-independent growth, and tumorigenesis in athymic mice. Cdk4-null fibroblasts proliferate at normal rates during early passages. WhereasCdk4+/+Ink4a/Arf−/− cells are immortal in culture,Cdk4−/−Ink4a/Arf−/− cells undergo senescence during continuous culture, as do wild-type cells. Activated Ras also induces premature senescence inCdk4−/−Ink4a/Arf−/− cells andCdk4−/− cells with DNp53 expression. Thus, Cdk4 deficiency causes senescence in a unique Arf/p53-independent manner, which accounts for the loss of transformation potential.Cdk4-null cells express high levels of p21Cip1/Waf1with increased protein stability. Suppression of p21Cip1/Waf1by small interfering RNA (siRNA), as well as expression of HPV-E7 oncoprotein, restores immortalization and Ras-mediated transformation in Cdk4−/−Ink4a/Arf−/− cells and Cdk4−/− cells with DNp53 expression. Therefore, Cdk4 is essential for immortalization, and suppression of Cdk4 could be a prospective strategy to recruit cells with inactive Arf/p53 pathway to senescence.