Protein kinase C (PKC)-mediated intracellular signaling participates in several key steps of hematopoietic cell differentiation. The epsilon isoform of PKC has been associated with erythroid differentiation as well as with the early phases of megakaryocytic (MK) lineage commitment. Here, we worked on the hypothesis that PKCepsilon expression levels might be modulated during MK differentiation, with a specific role in the early as well as in the late phases of thrombopoiesis. We demonstrate that--at variance with the erythroid lineage development--PKCepsilon is completely downmodulated in TPO-induced CD34 cells from day 6 onward. The forced expression of PKCepsilon in the late phases of MK differentiation delays the phenotypic differentiation of progenitors likely via Bcl-xL upregulation. Moreover, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), known as a negative regulator of early erythroid expansion, is not apoptogenic for thrombopoietin-induced CD34 cells, but rather accelerates their maturation. However, PKCepsilon levels negatively interfere also with the effects of TRAIL in MK differentiation. PKCepsilon can therefore be considered a signaling intermediate whose expression levels are finely tuned, with a virtually opposite kinetic, in erythroid versus megakaryocytic lineages, to adequately respond to the signaling requirements of the specific hematopoietic lineage.