Abstract A major operational problem for blood bank is supplying hospitals with platelets which have a short 5-day storage period. The high proliferative potential of cord blood hematopoietic stem-progenitor cells (CB HSPCs) permits the ex vivo expansion of megakaryocytes (MKs) for post-transplantation support and possibly the production of mature platelets for transfusion. MNCs from pooled CB samples (5) were enriched for CD34+ HSPCs using the StemSep Cell Separation System (StemCell Technologies). CD34+-enriched CB HSPCs were cultured in serum-free liquid culture medium supplemented with early-acting multilineage-specific recombinant human cytokines (25-100ng/mL of FL and SCF, and 5-20ng/mL of IL-3, IL-6 and G-CSF) and 50ng/mL of TPO, a MK-specific growth factor. After 25 days of culture, the minimum expansion of total nucleated cells was 100,000-fold in cultures supplemented with all 6 cytokines (at any assayed concentration). Approximately 80% and 50% of total cells were CD41+ and CD61+ respectively, corresponding to a 350,000 and 750,000-fold expansion respectively. Only 10% of cells were CD41+/42b+, CD61+/62+ or CD4+/15− (60,000 to 300,000-fold expansion). However, 50-70% of total cells expressed both CD41 (MK) and CD15 (granulocytes). The omission of IL-6 did not affect total or specific cell expansion. However, the absence of TPO, FL, SCF, IL-3 or G-CSF resulted in overall lower MK expansion due to a lower total cell expansion. In conclusion, extensive expansion of CB HSPCs can be obtained in ex vivo cultures. However, in the conditions used, TPO does not increase the frequency of MK progenitors nor induce the maturation of MKs.