The role of the marrow microenvironment in the pathophysiology of myelodysplastic syndromes (MDSs) remains controversial. Using stromal/hematopoietic cell cocultures, we investigated the effects of stroma-derived signals on apoptosis sensitivity in hematopoietic precursors. The leukemia-derived cell line KG1a is resistant to proapoptotic ligands. However, when cocultured with the human stromal cell line HS5 (derived from normal marrow) and exposed to tumor necrosis factor-α (TNF-α), KG1a cells showed caspase-3 activation and induction of apoptosis. Apoptosis was contact dependent. Identical results were obtained in coculture with primary stroma. Gene-expression profiling of KG1a cells identified coculture-induced up-regulation of various genes involved in apoptosis, including PYCARD. Suppression of PYCARD expression in KG1a by miRNA interfered with apoptosis. Knockdown of the TNF receptor 1 (TNFR1) or TNFR2 in HS5 cells had no effect. However, knockdown of R1 in KG1a cells prevented TNF-α–induced apoptosis, while apoptosis was still induced by TNF-α–related apoptosis-inducing ligand. Primary CD34⁺ cells from MDS marrow, when cocultured with HS5 and TNF-α, also underwent apoptosis. In contrast, no apoptosis was observed in CD34⁺ cells from the marrow of healthy donors. These data indicate that stroma may convey not only protective effects on hematopoietic cells, but, dependent upon the milieu, may also facilitate apoptosis.