Tumor necrosis factor (TNF)-alpha, a potent stimulus of nuclear factor-kappaB (NF-kappaB), is up-regulated in myelodysplastic syndrome (MDS). Here, we show that bone marrow mononuclear cells (BMMCs) and purified CD34+ cells from patients with low-grade/early-stage MDS (refractory anemia/refractory anemia with ring sideroblasts [RA/RARS]) have low levels of NF-kappaB activity in nuclear extracts comparable with normal marrow, while patients with RA with excess blasts (RAEB) show significantly increased levels of activity (P = .008). Exogenous TNF-alpha enhanced NF-kappaB nuclear translocation in MDS BMMCs above baseline levels. Treatment with arsenic trioxide (ATO; 2-200 microM) inhibited NF-kappaB activity in normal marrow, primary MDS, and ML1 cells, even in the presence of exogenous TNF-alpha (20 ng/mL), and down-regulated NF-kappaB-dependent antiapoptotic proteins, B-cell leukemia XL (Bcl-XL), Bcl-2, X-linked inhibitor of apoptosis (XIAP), and Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme (FLICE) inhibitory protein (FLIP), leading to apoptosis. However, overexpression of FLIP resulted in increased NF-kappaB activity and rendered ML1 cells resistant to ATO-induced apoptosis. These data are consistent with the observed up-regulation of FLIP and resistance to apoptosis with advanced MDS, where ATO as a single agent may show only limited efficacy. However, the data also suggest that combinations of ATO with agents that interfere with other pathways, such as FLIP autoamplification via NF-kappaB, may have considerable therapeutic activity.