Clonotypic elimination of activated T cells through Fas-Fas ligand (CD95-CD95L) interactions is one mechanism of peripheral self-tolerance. T cell receptor (TCR) stimuli trigger FasL synthesis but also sensitize activated T cells to Fas-mediated apoptosis through an unknown mechanism. Here we show that TCR restimulation of activated human CD4(+) T cells resulted in Fas translocation into lipid raft microdomains before binding FasL, rendering these cells sensitive to apoptosis after stimulation with bivalent antibody or FasL. Disruption of lipid rafts reduced sensitivity to Fas-mediated apoptosis after TCR restimulation. Thus, the redistribution of Fas and other tumor necrosis factor family receptors into and out of lipid rafts may dynamically regulate the efficiency and outcomes of signaling by these receptors.