The cyclinD:CDK4/6:Rb axis is dysregulated in a variety of human cancers. Targeting this pathway has proven to be a successful therapeutic approach in ER breast cancer. In this study, and preclinical breast cancer models were used to investigate the expanded use of the CDK4/6 inhibitor, abemaciclib. Using a panel of 44 breast cancer cell lines, differential sensitivity to abemaciclib was observed and was seen predominately in the luminal ER/HER2 and ER/HER2 subtypes. However, a subset of triple-negative breast cancer (TNBC) cell lines with intact Rb signaling were also found to be responsive. Equivalent levels of tumor growth inhibition were observed in ER/HER2, ER/HER2 as well as biomarker selected TNBC xenografts in response to abemaciclib. In addition, abemaciclib combined with hormonal blockade and/or HER2-targeted therapy induced significantly improved antitumor activity. CDK4/6 inhibition with abemaciclib combined with antimitotic agents, both and , did not antagonize the effect of either agent. Finally, we identified a set of Rb/E2F-regulated genes that consistently track with growth inhibitory response and constitute potential pharmacodynamic biomarkers of response to abemaciclib. Taken together, these data represent a comprehensive analysis of the preclinical activity of abemaciclib, used alone or in combination, in human breast cancer models. The subtypes most likely to respond to abemaciclib-based therapies can be identified by measurement of a specific set of biomarkers associated with increased dependency on cyclinD:CDK4/6:Rb signaling. These data support the clinical development of abemaciclib as monotherapy or as a combination partner in selected ER/HER2, HER2/ER, and TNBCs. .