Co-infusion of ex-vivo generated megakaryocytic progenitors with hematopoietic stem/progenitor cells (HSC/HPC) may contribute to a faster platelet recovery upon umbilical cord blood (UCB) transplantation. A two stage protocol containing cell expansion and megakaryocyte (Mk) differentiation was established using human UCB CD34+-enriched cells. The expansion stage used a pre-established protocol supported by a human bone marrow mesenchymal stem cells (MSC) feeder layer and the differentiation stage used TPO (100ng/mL) and IL-3 (10ng/mL). 18% of culture-derived Mks had higher DNA content (>4N) and were able to produce platelet-like particles. The proliferation extent of CD34+ cells obtained in the expansion stage (FI-CD34+), rather than expansion duration, determined as a key parameter for efficient megakaryocytic differentiation. A maximum efficiency yield (EY) of 48±7.7Mks/input CD34+ cells was obtained for a FI-CD34+ of 17±2.5, where a higher FI-CD34+ of 42±13 resulted in a less efficient megakaryocytic differentiation (EY of 22±6.7 and 19±4.6 %CD41).