Although the effect of thrombopoietin (TPO) on megakaryocyte production is well established, its role in the commitment of multipotential hematopoietic progenitors to the megakaryocytic lineage remains to be determined. In the present study, we attempted to clarify the determination process of megakaryocytic lineage as a terminal differentiation pathway under stimulation with TPO. Day 7 cultured cells grown by TPO derived from cord blood CD34⁺ cells were divided into four subpopulations on the basis of CD34 and CD41 expression. The CD34⁻/CD41⁻ cells showed the labeling pattern of anti-CD42b and anti-CD9 antibodies closer to that of the CD34⁺/CD41⁻ cells than the CD34⁺/CD41⁺ cells. Replating experiments revealed that approximately 40% of the CD34⁻/CD41⁻ cells proliferated in response to a combination of growth factors, and more than 80% of them were pure erythroid precursors. However, this subpopulation failed to grow/survive and fell into apoptosis in the presence of TPO alone. In contrast, the CD34⁺/CD41⁺ cells, which predominantly contained megakaryocytic precursors, exerted a low but significant proliferative potential in the presence of TPO. The insufficient response to TPO of the CD34⁻/CD41⁻ cells may result from the apparently low expression of c-Mpl, as determined by flow cytometric analysis and reverse transcription-polymerase chain reaction analysis. Therefore, these results suggest that the apoptosis of hematopoietic precursors other than megakaryocytic precursors is related to the determination of the terminal differentiation under the influence of TPO.