Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potentially useful anticancer agent with exquisite selectivity for cancer cells. Unfortunately, many cancers exhibit or acquire resistance to TRAIL. We report herein that TRAIL activates a TGF-ß-activated kinase 1→mitogen-activated protein kinase (MAPK) kinase 3 (MKK3)/MKK6→p38 pathway in prostate cancer cells that transcriptionally upregulates expression of the antiapoptotic BCL-2 family member MCL-1. TRAIL alone triggered robust formation of the "death-inducing signaling complex", activation of the initiator caspase-8, and truncation of the BH3-only protein BID (tBID). Nevertheless, simultaneous disruption of the p38 MAPK pathway was required to suppress MCL-1 expression, thereby allowing tBID to activate the proapoptotic BCL-2 family member BAK and stimulate mitochondrial outer membrane permeabilization (MOMP). Release of the inhibitor-of-apoptosis antagonist, Smac/DIABLO, from the intermembrane space was sufficient to promote TRAIL-induced apoptosis, whereas release of cytochrome c and apoptosome function were dispensable. Even following MOMP, however, mitochondrial-generated reactive oxygen species activated a secondary signaling pathway, involving c-Jun N-terminal kinases, that likewise upregulated MCL-1 expression and partially rescued cells from death. Thus, stress kinases activated at distinct steps in the extrinsic pathway mediate TRAIL resistance through maintenance of MCL-1 expression. / text