Actin cytoskeleton has been shown to play a regulating role in several signaling pathways, and disruption of actin filament has been reported to increase sTNF-alpha-induced cell death. However, whether actin is involved in tmTNF-alpha-mediated cytotoxicity remains unclear. Here, we demonstrated that pretreatment of HL-60 with CytD or LatA to depolymerize actin significantly suppressed tmTNF-alpha-mediated apoptosis. Interestingly, tmTNF-alpha increased the actin immunoprecipitated by anti-TNFR2 but not anti-TNFR1 antibody, and disruption of the actin filament totally blocked this effect. In addition, TNFR1 knockdown by siRNA did not affect tmTNF-alpha-mediated cytotoxicity and the inhibitory effect of CytD, suggesting that the involvement of actin in the tmTNF-alpha-induced apoptosis is linked to the TNFR2 pathway. Our results revealed further that tmTNF-alpha signaled the inhibition of I kappa B degradation and NF-kappa B activity by recruiting RIP1 to and uncoupling TRAF2 from the TNFR2 complex. Nevertheless, CytD totally reversed the tmTNF-alpha signaling and activated NF-kappa B by recruiting TRAF2 to and dissociating RIP1 from the TNFR2 complex. Furthermore, tmTNF-alpha led to activation of caspase-8 by dissociation of cFLIP from TNFR2 and inhibition of the cFLIP expression. Activated caspase-8 cleft RIP1 to suppress NF-kappa B activity and also mediated tmTNF-alpha-induced apoptosis. However, CytD blocked the tmTNF-alpha-induced uncoupling of cFLIP from TNFR2 and prevented caspase-8 activation and the resulting cleavage of RIP1, converting the signaling for tmTNF-alpha-mediated apoptosis into one for activating NF-kappa B to survive. These results suggest that the actin cytoskeleton functions in transmitting signals via TNFR2 to mediate tmTNF-alpha-induced apoptosis. J. Leukoc. Biol. 89: 917-926; 2011.