Previous studies suggest that cells within the CD34⁺ hematopoietic stem cell compartment are endowed with immune regulatory activity. Furthermore, it is possible to expand the human regulatory cells upon short-term culture of purified CD34⁺ cells with an early-acting cytokine cocktail. We now show that addition of anti-CD28, anti-CD2, interleukin-2 (IL-2), anti–IL-10, or IL-12 to the bulk mixed lymphocyte reaction (MLR) cannot reverse the inhibitory activity of the CD34⁺ cells, ruling out anergy-based mechanisms or mechanisms involving Th1-Th2 skewing. Furthermore, phenotyping of cells present after addition of CD34⁺ cells to the bulk MLR ruled out potential induction of plasmacytoid dendritic precursors, known to be endowed with regulatory activity. In contrast, the inhibitory activity of CD34⁺ cells could be reversed by adding the caspase inhibitor BD-FMK to the bulk MLR, indicating a deletion-based mechanism. The deletion can be inhibited by anti–tumor necrosis factor-α (anti–TNF-α) and not by anti–transforming growth factor-β (anti–TGF-β), suggesting a potential role for TNF-α in the regulatory activity of CD34⁺ cells.