We have studied the effects of recombinant human thrombopoietin (TPO; mpl ligand) on the proliferation of human primitive hematopoietic progenitors in vitro. CD34⁺ cells were enriched for cell-cycle-dormant primitive progenitors by separation on the basis of expression of c-kit and CD38. In the presence of varying combinations of TPO, Steel factor (SF), and interleukin-3 (IL-3), CD34⁺/c-kit^low/CD38^neg/low cells produced fewer colonies than CD34⁺/c-kit^low/CD38^high cells. However, when cultured in suspension for 7 days and re-plated in methylcellulose culture for measurement of colony-forming cells, the former population generated more colony-forming cells than the latter. In suspension culture of CD34⁺/c-kit^low/CD38^neg/low cells, TPO acted synergistically with SF and/or IL-3 in support of the production of colony-forming cells for granulocyte/macrophage colonies, erythroid colonies, and mixed colonies. Culture studies of individual CD34⁺/c-kit^low/CD38^neg/low cells provided the evidence for the direct nature of the effects of TPO. When combined with SF, TPO showed stronger stimulation of production of progenitors in suspension culture than other early-acting factors, such as IL-6, IL-11, and granulocyte colony-stimulating factor (G-CSF). TPO may be an important cytokine for in vitro manipulation of human hematopoietic stem cells.