Cell cycle progression from G₁ to S phase depends on phosphorylation of pRb by complexes containing a cyclin (D type or E type) and cyclin-dependent kinase (e.g., cdk2, cdk4, or cdk6). Ink4 proteins function to oppose the action of cdk4/6-cyclin D complexes by inhibiting cdk4/6. We employed genetic and pharmacologic approaches to study the interplay among Ink4 proteins and cdk4/6 activity in vivo. Mouse embryo fibroblasts (MEF) lacking p16^Ink4a and p18^Ink4c showed similar growth kinetics as wild-type MEFs despite increased cdk4 activity. In vivo, germline deficiency of p16^Ink4a and p18^Ink4c resulted in increased proliferation in the intermediate pituitary and pancreatic islets of adult mice, and survival of p16^Ink4a−/−;p18^Ink4c−/− mice was significantly reduced due to aggressive pituitary tumors. Compensation among the Ink4 proteins was observed both in vivo in p18^Ink4c−/− mice and in MEFs from p16^Ink4a−/−, p18^Ink4c−/−, or p16^Ink4a−/−;p18^Ink4c−/− mice. Treatment with PD 0332991, a specific cdk4/6 kinase inhibitor, abrogated proliferation in those compartments where Ink4 deficiency was associated with enhanced proliferation (i.e., islets, pituitary, and B lymphocytes) but had no effect on proliferation in other tissues such as the small bowel. These data suggest that p16^Ink4a and p18^Ink4c coordinately regulate the in vivo catalytic activity of cdk4/6 in specific compartments of adult mice. [Cancer Res 2007;67(10):4732–41]