Recently, several lines of evidence have indicated an expanded role for thrombopoietin (TPO) and its receptor, c-mpl, in hematopoiesis. In addition to being the primary physiological regulator of platelet production, it is now apparent that TPO also acts during early hematopoiesis. To futher define the role of TPO in early hematopoiesis we have identified discrete murine and human stem cell populations with respect to c-mpl expression and evaluated their potential for hematopoietic engraftment. Fluorescence-activated cell sorter analysis of enriched stem cell populations showed the presence of c-mpl expressing subpopulations. Approximately 50% of the murine fetal liver stem cell–enriched population, AA4⁺Sca⁺c-kit⁺, expressed c-mpl. Analysis of the murine marrow stem cell population Lin^loSca⁺c-kit⁺ showed that 70% of this population expressed c-mpl. Expression of c-mpl was also detected within the human bone marrow CD34⁺CD38⁻ stem cell progenitor pool and approximately 70% of that population expressed c-mpl. To rigorously evaluate the role of TPO/c-mpl in early hematopoiesis we compared the repopulation capacity of murine stem cell populations with respect to c-mpl expression in a competitive repopulation assay. When comparing the fetal liver progenitor populations, AA4⁺Sca⁺c-kit⁺c-mpl⁺and AA4⁺Sca⁺c-kit⁺c-mpl⁻, we found that stem cell activity segregates with c-mpl expression. This result is complemented by the observation that the Lin^loSca⁺ population of c-mplgene-deficient mice was sevenfold less potent than Lin^loSca⁺ cells from wild-type mice in repopulating activity. The engraftment potential of the human CD34⁺CD38⁻c-mpl⁺ population was evaluated in a severe combined immunodeficient-human bone model. In comparison to the CD34⁺CD38⁻c-mpl⁻ population, the CD34⁺CD38⁻c-mpl⁺ cells showed significantly better engraftment. These results demonstrate a physiological role for TPO and its receptor, c-mpl, in regulating early hematopoiesis.