Whereas maintenance of hematopoietic stem cells (HSCs) is a requisite for life, uncontrolled expansion of HSCs might enhance the propensity for leukemic transformation. Accordingly, HSC numbers are tightly regulated. The identification of physical cellular HSC niches has underscored the importance of extrinsic regulators of HSC homeostasis. However, whereas extrinsic positive regulators of HSCs have been identified, opposing extrinsic repressors of HSC expansion in vivo have yet to be described. Like many other acute and chronic inflammatory diseases, bone marrow (BM) failure syndromes are associated with tumor necrosis factor-α (TNF) overexpression. However, the in vivo relevance of TNF in the regulation of HSCs has remained unclear. Of considerable relevance for normal hematopoiesis and in particular BM failure syndromes, we herein demonstrate that TNF is a cell-extrinsic and potent endogenous suppressor of normal HSC activity in vivo in mice. These effects of TNF involve two distinct TNF receptors.