Objective: This study was performed to determine the immunophenotype of Megakaryocyte progenitor cells differentiated from UCB CD133+ and CD133− cells under the effects of interleukin–3 (IL–3), interleukin–6 (IL–6), stem cell factor (SCF) and thrombopoietin (TPO) in vitro. Materials and Methods: CD133+and CD133− cells were isolated by using CD133 isolation kit following the manufacturer’s instructions. Then, they were seeded in liquid serum free expansion mediums supplemented with the cytokine cocktail including IL–3, IL–6, SCF and TPO. The expression rate of CD34, CD41, CD61 and CD42b were measured on the days 0 and 7 of culture using flow cytometry. Student’s t–test was used for the comparisons and a p value less than 0.05 was considered to be significant. Results: Expressions of megakaryocytic markers on CD133+ cells were always higher than CD133− cells. CD133+ cells have higher potential of generating Mk colonies in vitro. Conclusion: CD133+ subset may be used as an alternative source for Mk progenitor cells production and these cells may improve platelet recovery after UCB transplantation.