Histone deacetylase inhibitor (HDACI)–induced thrombocytopenia (TCP) is a major dose-limiting toxicity of this new class of drugs. Using preclinical models to study the molecular and biologic events that underpin this effect of HDACI, we found that C57BL/6 mice treated with both the HDAC1/2-selective HDACI romidepsin and the pan-HDACI panobinostat developed significant TCP. HDACI-induced TCP was not due to myelosuppression or reduced platelet lifespan, but to decreased platelet release from megakaryocytes. Cultured primary murine megakaryocytes showed reductions in proplatelet extensions after HDACI exposure and a dose-dependent increase in the phosphorylation of myosin light chain 2 (MLC2). Phosphorylation of MLC to phospho-MLC (pMLC) and subsequent proplatelet formation in megakaryocytes is regulated by the Rho-GTPase proteins Rac1, CDC42, and RhoA. Primary mouse megakaryocytes and the human megakaryoblastic cell line Meg-01 showed reductions in Rac1, CDC42, and RhoA protein levels after treatment with HDACIs. We were able to overcome HDACI-induced TCP by administering the mouse-specific thrombopoietin (TPO) mimetic AMP-4, which improved platelet numbers to levels similar to untreated controls. Our report provides the first detailed account of the molecular and biologic processes involved in HDACI-mediated TCP. Moreover, our preclinical studies provide evidence that dose-limiting TCP induced by HDACIs may be circumvented using a TPO mimetic.