Abstract It is well established that phosphorylation of Rb-family pocket proteins by CDK4 and CDK6 is important for the commitment of cancer cells to a new cell cycle and the initiation of the G1-S phase transition. Abemaciclib is a potent inhibitor of the kinase activity of both CDK4 and CDK6 and is currently undergoing clinical testing. To better understand the molecular determinants of response to abemaciclib, we tested its anti-proliferative activity across a panel of over 500 well characterized cancer cell lines. Statistical approaches were employed to uncover genomic features associated with the response. Candidate markers of sensitivity and resistance were further tested by genetic manipulations in vitro. In vivo models representing the candidate molecular marker of sensitivity were identified and drug efficacy examined. Three broad classes of response were identified. The class of tumors cells most resistant to abemaciclib showed enrichment for RB1 mutations. Conversely, cell lines with amplification of CCND2 and CCND3 were among the very most sensitive tumor cells and tumor cells with these markers showed evidence of senescence and apoptosis after either depletion of the cognate D-cyclin or treatment with abemaciclib. In vivo models of tumors harboring CCND2 and CCND3 gene amplification were very sensitive to abemaciclib treatment and showed evidence of tumor regression. Citation Format: Xueqian Gong, Li-Chun Chio, MaryJo Lallena, Farhana Merzoug, Shaoyou Chu, Yue Webster, Jack Dempsey, Xiwen Ma, Alfonso De Dios, Richard Beckman, Sean G. Buchanan. Molecular features that determine the sensitivity of cancer cells to abemaciclib, an inhibitor of CDK4 and CDK6. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3104. doi:10.1158/1538-7445.AM2015-3104