Small molecule inhibitors of cyclin dependent kinases 4 and 6 (CDK4/6) were approved for the treatment of cancer on the basis of their tumor-intrinsic cytostatic activity. However, the emerging role of these inhibitors as immunomodulatory agents is less understood. Using integrated single cell transcriptomic and proteomic analyses, we demonstrated that CDK4/6 inhibition promotes the formation of phenotypic and functional T cell memory. Short-term priming with a CDK4/6 inhibitor promoted long-term endogenous anti-tumor T cell immunity in mice, enhanced the persistence and therapeutic efficacy of chimeric antigen receptor (CAR)-T cells, and induced an T cell phenotype that is optimal for targeting with immune checkpoint blockade in both mice and melanoma patients. Taken together, these mechanistic insights uncover a novel pathway governing T cell memory formation, and significantly broaden the prospective utility of CDK4/6 inhibitors as clinical tools to boost anti-tumor T cell immunity.