In Fanconi anemia (FA) C mice tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) have key roles in the pathogenesis of bone marrow failure. In FA subjects TNF-α was found to be increased in the serum and overproduced by patient-derived B-cell lines. In acquired aplastic anemia, a disease in which, similarly to FA, marrow failure occurs, TNF-α and IFN-γ act as late mediators of the stem cell damage and are overexpressed in patient marrow lymphocytes. This study evaluated in marrow mononuclear cells (MNCs) of patients with FA, the expression of negative modulators of the hematopoiesis, such as TNF-α, IFN-γ, macrophage inflammatory protein 1α (MIP-1α), and surface Fas ligand, and the role of TNF-α on FA erythropoiesis in vitro. TNF-α and IFN-γ were significantly overexpressed in stimulated marrow MNCs of FA patients as compared to healthy controls. MIP-1α and Fas ligand were undetectable in patients and controls. In bone marrow cultures, the addition of anti–TNF-α increased the size and significantly increased the number of erythroid colony-forming units and erythroid burst-forming units grown from FA patients but not from healthy controls. This indicates that FA subjects have a marrow TNF-α activity that inhibits erythropoiesis in vitro. TNF-α has a relevant role in the pathogenesis of erythroid failure in FA patients.