Objective— We previously determined that protein kinase C δ (PKCδ) regulates platelet function. However, the function of PKCδ in megakaryopoiesis is unknown. Approach and Results— Using PKCδ ^-/- and wild-type littermate mice, we found that deficiency of PKCδ caused an increase in white blood cells and platelet counts, as well as in bone marrow and splenic megakaryocytes ( P <0.05). Additionally, the megakaryocyte number and DNA content were enhanced in PKCδ ^-/- mouse bone marrow after culturing with exogenous thrombopoietin compared with wild-type ( P <0.05). Importantly, thrombopoietin-induced signaling was also altered with PKCδ deletion because both extracellular signal-regulated kinase and Akt308 phosphorylation were heightened in PKCδ ^-/- megakaryocytes compared with wild-type. Finally, PKCδ ^-/- mice recovered faster and had a heightened rebound thrombocytosis after thrombocytopenic challenge. Conclusions— These data suggest that PKCδ is an important megakaryopoietic protein, which regulates signaling induced by thrombopoietin and represents a potential therapeutic target.