Abstract Objective To evaluate the megakaryocyte potential of normal bone marrow (NBM) CD34 +CD133 + cells, a subset offering a possible alternative for clinical CD34 immunoselection, we evaluated their colony-forming unit megakaryocyte (CFU-Mk) content and their ability to produce clonogenic Mk progenitors in comparison with the CD133 − subset. Materials and Methods Sorted NBM CD34 +CD133 + and CD34 +CD133 − subsets were evaluated for Mk clonogenic capacity before and after in vitro proliferation in serum-free liquid culture containing kit ligand, Flt3 ligand, thrombopoietin, interleukin-3, and interleukin-6. The segregation of CFU-Mk according to the expression of CD34, CD133, and CD41 was compared between fresh BM cells and expanded cells. Results Although the fresh NBM CD133 −CD34 + subset included two thirds CFU-Mk, only the CD133 + subset contained primitive cells able to produce all categories of CFU-Mk in vitro. Immunophenotyping confirmed that CD41 antigen is nonspecific for Mk lineage and showed that the usual CD34 +CD41 + subset does not specifically define a CFU-Mk population. The segregation of CFU-Mk before and after expansion according to CD34, CD41, or CD133 was modified in relation with down-regulation of CD34 and CD133 antigens and up-regulation of CD41 antigen. Conclusion The NBM CD133 + subset contains primitive cells able to generate CFU-Mk, a subset probably relevant to platelet recovery after infusion. The alteration of antigen expression during in vitro proliferation calls for caution in the identification of the different categories of Mk subsets produced and in the assessment of their predictivity for in vivo platelet production.