Maintenance of hematopoietic stem cells (HSCs) depends on interaction with their niche. Here we show that the long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, MPL, are a quiescent population in adult bone marrow (BM) and are closely associated with THPO-producing osteoblastic cells. THPO/MPL signaling upregulated β1-integrin and cyclin-dependent kinase inhibitors in HSCs. Furthermore, inhibition and stimulation of THPO/MPL pathway by treatments with anti-MPL neutralizing antibody, AMM2, and with THPO showed reciprocal regulation of quiescence of LT-HSC. AMM2 treatment reduced the number of quiescent LT-HSCs and allowed exogenous HSC engraftment without irradiation. By contrast, exogenous THPO transiently increased quiescent HSC population and subsequently induced HSC proliferation in vivo. Altogether, these observations suggest that THPO/MPL signaling plays a critical role of LT-HSC regulation in the osteoblastic niche.