Tumor cell proliferation is frequently associated with genetic or epigenetic alterations in key cell cycle molecules that regulate the activity of cyclin-dependent kinases (CDKs). These protein kinases control the progression through the different phases of the cell division cycle. Tumor-associated alterations in their activating partners, cyclins, or in CDK inhibitors help to sustain proliferation with independence from external mitogenic or anti-mitogenic signals. The significant frequency of these alterations in human cancer and recent studies in genetically-engineered mouse models predict that inhibition of CDKs might have therapeutic value. Most interphase CDKs are dispensable for mouse development but their inhibition may prevent DNA replication in some specific tumor cells. On the other hand, inactivation of mitotic CDKs efficiently prevents progression throughout the mitotic cell cycle. The combination of biochemical and genetic data on the function of these regulators will be instrumental to define new targeted therapies for inhibiting proliferation in cancer cells.