Background: The combination of IL-1, IL-6, IL-11, stem cell factor (SCF), and thrombopoietin (TPO) is capable of stimulating megakaryocytopoiesis using a bone marrow CD34+ cell population in a 10- to 12-day expansion culture. We evaluated the ex vivo effect of these cytokines in combination with flt-3 ligand (FL) in expanding CD34+ umbilical cord blood (UCB) cells. Material and Methods: To investigate the developmental kinetics of cell expansion, CD34+ cells were selected from 19 UCB units and cultured for 5–21 days in 24-well plates in serum-containing medium (1×104 cells/ml). Cytokines included IL-1 (10 ng/ml), IL-6, IL-11, and SCF (each 25 ng/ml) as a basic cocktail with the addition of either TPO (50 ng/ml) or FL (50 ng/ml) or both TPO and FL. CD34 and CD41 antibodies were used to identify progenitors and megacaryocytic (MK) cells by flow cytometry, and CFU-MK assay was performed to quantify MK progenitors. Results: The medium containing TPO plus FL provided the best stimulus for developing MK cells and resulted in a significant expansion of CD41+ cells from day 5 to day 11, with a mean increase of up to 123-fold (14- to 284-fold) in contrast to 78-fold expansion of total nucleated cells (NC) (12- to 150-fold, p < 0.01, n = 8). The percentage of CD34+ cells showed a significant decrease from 83% to less than 1%, as well as the relative concentration of MK progenitor cells which dropped from a mean of 128 per 104 CD34+ cells to 10–70 at day 5. However, because of NC amplification, a slight expansion in CFU-MK could be observed showing a 7.8-fold increase using all six cytokines over 8 days. Conclusion: UCB CD34+ cell expansion over 14 days by using the above-mentioned cytokine combinations induces a strong maturation pressure and causes the progenitors to undergo MK differentiation. Finally, it results in only a slight expansion of MK progenitor cells measured by the CFU-MK assay.