In acute myeloid leukemia (AML), resistance to chemotherapy is associated with defects in both the extrinsic and intrinsic pathways of apoptosis. Novel agents that activate endogenous apoptosis-inducing mechanisms directly may be potentially useful to overcome chemoresistance in AML. We examined the mechanisms of apoptosis induction by the novel synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) in AML cells. CDDO-induced apoptosis was associated with the loss of mitochondrial inner transmembrane potential, caspases activation, the translocation of apoptosis-inducing factor to the nucleus, and DNA fragmentation in AML cells. Apoptosis was equally evident in cells deficient in caspase-9 or caspase-8 after exposure to CDDO, suggesting caspase-independent cell death. The use of small interfering RNA to reduce the expression of apoptosis-inducing factor partially inhibited CDDO-induced apoptosis in AML cells. Cells overexpressing Bcl-2 were markedly resistant to CDDO-induced apoptosis. Moreover, CDDO promoted the release of cytochrome c from isolated mitochondria, suggesting that CDDO targets the mitochondria directly to trigger the intrinsic pathway of cell death in intact cells. Together, these results suggest that CDDO functions by activating the intrinsic pathway of apoptosis and initiates caspase-dependent and independent cell death. The direct modulation of mitochondrial-mediated, caspase-independent apoptosis by CDDO may be advantageous for overcoming chemoresistance in AML.