A large number of human cancers display alterations in the Ink4a/cyclin D/Cdk4 genetic pathway, suggesting that activation of Cdk4 plays an important role in oncogenesis. Here we report that Cdk4-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. Whereas Cdk4(+/+)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 in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) 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 p21(Cip1/Waf1) with increased protein stability. Suppression of p21(Cip1/Waf1) by 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.