Multiple lines of evidence indicate that thrombopoietin (TPO) contributes to the development of hematopoietic stem cells (HSC), supporting their survival and proliferation in vitro. To determine whether TPO supports the impressive expansion of HSC observed following transplantation, we transplanted normal marrow cells into lethally irradiated Tpo^–/– and Tpo^+/+ mice and quantified HSC self-renewal and expansion and hematopoietic progenitor cell homing. Although essentially identical numbers of marrow-associated colony forming unit–culture (a surrogate measure of stem cell homing) were observed in each type of recipient 24 hours following transplantation, we found that a minimum of fourfold greater numbers of marrow cells were required to radioprotect Tpo-null mice than to radioprotect controls. To assess whether long-term repopulating (LTR) HSCs self-renew and expand in Tpo^–/– recipients or controls, we performed limiting-dilution secondary transplants using donor cells from the Tpo^–/– or Tpo^+/+ recipients 5–7.5 weeks following primary transplantation. We found that LTR HSCs expand to levels 10–20 times greater within this time period in normal recipients than in Tpo-null mice and that physiologically relevant amounts of TPO administered to the Tpo^–/– recipients could substantially correct this defect. Our results establish that TPO greatly promotes the self-renewal and expansion of HSCs in vivo following marrow transplantation.