We investigated the physiological role of endogenous MAPK-activating death domain-containing protein (MADD), a splice variant of the IG20 gene, that can interact with TNFR1 in tumor necrosis factor-alpha (TNFalpha)-induced activation of NF-kappaB, MAPK, ERK1/2, JNK, and p38. Using exon-specific short hairpin RNAs expressing lentiviruses, we knocked down the expression of all IG20 splice variants or MADD, which is overexpressed in cancer cells. Abrogation of MADD expression rendered cells highly susceptible to TNFalpha-induced apoptosis in the absence of cycloheximide. It also resulted in a dramatic loss in TNFalpha-induced activation of MAPK without any apparent effect on NF-kappaB activation. This observation was substantiated by an accompanying loss in the activation of p90RSK, a key downstream target of MAPK, whereas the NF-kappaB-regulated interleukin 6 levels remained unaffected. Endogenous MADD knockdown, however, did not affect epidermal growth factor-induced MAPK activation thereby demonstrating the specific requirement of MADD for TNF receptor-mediated MAPK activation. Re-expression of short hairpin RNA-resistant MADD in the absence of endogenous IG20 expression rescued the cells from TNFalpha-induced apoptosis. The requirement for MADD was highly specific for TNFalpha-induced activation of MAPK but not the related JNK and p38 kinases. Loss of MADD expression resulted in reduced Grb2 and Sos1/2 recruitment to the TNFR1 complex and decreased Ras and MEKK1/2 activation. These results demonstrate the essential role of MADD in protecting cancer cells from TNFalpha-induced apoptosis by specifically activating MAPKs through Grb2 and Sos1/2 recruitment, and its potential as a novel cancer therapeutic target.