Our goal is to produce ex vivo-expanded human megakaryocytes (MK) cells from peripheral blood progenitor cell (PBPC) harvests for use in supplementing conventional autografts. In this paper we show the megakaryocytopoietic productivity of small-scale in vitro serum-free cultures of human CD34⁺ cells containing MK growth and development factor (MGDF) and stem cell factor (Kit ligand; SCF) ± granulocyte colony-stimulating factor (G-CSF). Cultures were characterized after 3, 6, 9, and 13 days by flow cytometry and clonogenic assays. CD34⁺ cells expanded 5.2- and 3.4-fold, and produced 2.2 and 2.4 CD34⁺/41⁺ cells per seeded CD34⁺ cell after 6 and 9 days in culture, respectively. None were detected at day 13. CD41⁺ cells expanded exponentially over 13 days. Colony-forming unit-megakaryocyte (CFU-MK) also expanded exponentially, but the proportion of the most primitive CFU-MK dropped from 45% to 1.5% and to <1% after 6 and 9 days, respectively. G-CSF increased total cell expansion, but decreased CD41⁺ frequency, yielding no gain in MK production. We also found that PB CD34⁺ cells cultured for 3-6 days are richer in primitive MK progenitors, while those cultured for 9-13 days have greater numbers of more differentiated MKs. Overall, the combination of MGDF+SCF proved sufficient for expanding CD34⁺/CD41⁺ cells. As the stage of ex vivo MK differentiation most conducive to optimal platelet production in vivo is not known, we are planning a clinical trial to determine the efficacy of ex vivo-expanded MKs on platelet recovery in relation to MK maturity.