The role of cytokines in regulation of hematopoietic stem cells (HSCs) remains poorly understood. Herein we demonstrate that thrombopoietin (THPO) and its receptor, MPL, are critically involved in postnatal steady-state HSC maintenance, reflected in a 150-fold reduction of HSCs in adult Thpo^−/− mice. Further, whereas THPO and MPL proved not required for fetal HSC expansion, HSC expansion posttransplantation was highly MPL and THPO dependent. The distinct role of THPO in postnatal HSC maintenance is accompanied by accelerated HSC cell-cycle kinetics in Thpo^−/− mice and reduced expression of the cyclin-dependent kinase inhibitors p57^Kip2 and p19^INK4D as well as multiple Hox transcription factors. Although also predicted to be an HSC viability factor, BCL2 failed to rescue the HSC deficiency of Thpo^−/− mice. Thus, THPO regulates posttransplantation HSC expansion as well as the maintenance of adult quiescent HSCs, of critical importance to avoid postnatal HSC exhaustion.