Inhibitors of cyclin-dependent kinases (CDKs) 4/6 have emerged as a powerful class of agents with clinical activity in several malignancies. Targeting the cell cycle represents a core attack on a defining feature of cancer. However, the mechanisms of action of agents selectively targeting CDK4/6 have few parallels in the current pharmaceutical armamentarium against cancer. Notably, CDK4/6 inhibitors act downstream of most mitogenic signaling cascades, and this has implications for both clinical efficacy and resistance. Core knowledge of cell-cycle processes has provided insights into the mechanisms of intrinsic resistance to CDK4/6 inhibitors; however, the basis of acquired resistance versus durable response is only beginning to emerge. This review focuses on the mechanism of action of CDK4/6 inhibitors as well as on biomarkers to direct their precision use in rationally developed combination therapies.