Self-renewal of hematopoietic stem cells (HSCs) is key to their reconstituting ability, but the factors regulating the process remain poorly understood. Here, we show that Interleukin-10 (IL-10), a pleiotropic immune modulating cytokine, can also play a role in regulating HSC self-renewal. First, a quantitative decrease of primitive hematopoietic cell populations, but not more matured cells, was observed in the bone marrows of IL-10 disrupted mice as determined by long-term in vitro cultures or in vivo competitive repopulation assays. In contrast, normal HSCs from 5-fluorouracil treated marrows cultured on the IL-10 secreting stroma displayed an enhanced repopulating activity compared with cells grown on control stroma, with ninefold higher numbers of donor-derived HSCs in the reconstituted recipient marrows. Moreover, limiting dilution transplantation assay demonstrated that exogenous addition of IL-10 in the stroma-free cultures of purified Lin- Sca-1+ c-kit+ cells caused three- to fourfold higher frequencies of HSCs in the 5-day short-term culture without indirect inhibitory effect of IL-10 on tumor necrosis factor-alpha or interferon-gamma secretion. Interestingly, primitive hematopoietic cells, including Lin- Sca-1+ c-kit+ or side population cells, expressed the surface receptor for IL-10, and microenvironmental production of IL-10 was sharply increased in the osteoblasts lining the trabecular regions of the radiation-stressed marrow but not in the steady-state marrows. These results show that IL-10 may be a ligand that can stimulate self-renewal of HSCs to promote their regeneration in addition to being a ligand for immune regulation. Disclosure of potential conflicts of interest is found at the end of this article.