Abstract The INK4 and CIP cyclin-dependent kinase (Cdk) inhibitors (CKI) activate pocket protein function by suppressing Cdk4 and Cdk2, respectively. Although these inhibitors are lost in tumors, deletion of individual CKIs results in modest proliferation defects in murine models. We have evaluated cooperativity between loss of all INK4 family members (using cdk4r24c mutant alleles that confer resistant to INK4 inhibitors) and p21Waf1/Cip1 in senescence and transformation of mouse embryo fibroblasts (MEF). We show that mutant cdk4r24c and p21 loss cooperate in pRb inactivation and MEF immortalization. Our studies suggest that cdk4r24c mediates resistance to p15INK4B/p16INK4A that accumulates over passage, whereas loss of p21 suppresses hyperoxia-induced Cdk2 inhibition and pRb dephosphorylation on MEF explantation in culture. Although cdk4r24c and p21 loss cooperate in H-rasV12/c-myc–induced foci formation, they are insufficient for oncogene-induced anchorage-independent growth. Interestingly, p21−/−; cdk4r24c MEFs expressing H-rasV12 and c-myc display detachment-induced apoptosis and are transformed by c-myc, H-rasV12, and Bcl-2. We conclude that the INK4 family and p21 loss cooperate in promoting pRb inactivation, cell immortalization, and H-rasV12/c-myc–induced loss of contact inhibition. In addition, absence of pRb function renders H-rasV12 + c-myc–transduced fibroblasts prone to apoptosis when deprived of the extracellular matrix, and oncogene-induced anchorage-independent growth of pocket protein–deficient cells requires apoptotic suppression. [Cancer Res 2007;67(9):4130–7]