Fas/Apo1 and other cytotoxic receptors of the tumor necrosis factor receptor (TNFR) family contain a cytoplasmic death domain (DD) [1–11] that activates the apoptotic process by interacting with the DD-containing adaptor proteins TNFR-associated DD protein (TRADD) [12,13] and Fas-associated DD protein (FADD/MORT1) [14,15], leading to the activation of cysteine proteases of the caspase family [16]. Stimulation of Fas/Apo1 leads to the formation of a receptor-bound death-inducing signaling complex (DISC), consisting of FADD and two different forms of caspase-8 [17–19]. Transient expression of a dominant-negative mutant of FADD impairs TNFR60-mediated and Fas/Apo1-mediated apoptosis [13,20], but has no effect on TNF-related apoptosis-inducing ligand (TRAIL/Apo2L)-induced cell death [7–10,21]. To study the function of FADD in DD-receptor signaling in more detail, we established HeLa cells that stably expressed a green fluorescent protein (GFP)-tagged dominant-negative mutant of FADD, GFP-ΔFADD. Interestingly, expression of this mutant inhibited cell death induced by TNFR60, Fas/Apo1 and TRAIL-R/Apo2. In addition, GFP-ΔFADD did not interfere with TNF-mediated gene induction or with activation of NF-κB or Jun N-terminal kinase (JNK), demonstrating that FADD is part of the TNFR60-initiated apoptotic pathway but does not play a role in TNFR60-mediated gene induction. Fas/Apo1-mediated activation of JNK was unaffected by the expression of GFP-ΔFADD, suggesting that in Fas/Apo1 signaling the apoptotic pathway and the activation of JNK diverge at a level proximal to the receptor, upstream of or parallel to FADD.