Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) acts as an apoptosis inducer for cancer cells sparing non-tumor cell targets. However, several phase I/II clinical trials have shown limited benefits of this molecule. In the present work, we investigated whether cell susceptibility to TRAIL ligation could be due to the presence of TRAIL death receptors (DRs) 4 and 5 in membrane microdomains called lipid rafts. We performed a series of analyses, either by biochemical methods or fluorescence resonance energy transfer (FRET) technique, on normal cells (i.e. lymphocytes, fibroblasts, endothelial cells), on a panel of human cancer B-cell lines as well as on CD19(+) lymphocytes from patients with B-chronic lymphocytic leukemia, treated with different TRAIL ligands, that is, recombinant soluble TRAIL, specific agonistic antibodies to DR4 and DR5, or CD34(+) TRAIL-armed cells. Irrespective to the expression levels of DRs, a molecular interaction between ganglioside GM3, abundant in lymphoid cells, and DR4 was detected. This association was negligible in all non-transformed cells and was strictly related to TRAIL susceptibility of cancer cells. Interestingly, lipid raft disruptor methyl-beta-cyclodextrin abrogated this susceptibility, whereas the chemotherapic drug perifosine, which induced the recruitment of TRAIL into lipid microdomains, improved TRAIL-induced apoptosis. Accordingly, in ex vivo samples from patients with B-chronic lymphocytic leukemia, the constitutive embedding of DR4 in lipid microdomains was associated per se with cell death susceptibility, whereas its exclusion was associated with TRAIL resistance. These results provide a key mechanism for TRAIL sensitivity in B-cell malignances: the association, within lipid microdomains, of DR4 but not DR5, with a specific ganglioside, that is the monosialoganglioside GM3. On these bases we suggest that lipid microdomains could exert a catalytic role for DR4-mediated cell death and that an ex vivo quantitative FRET analysis could be predictive of cancer cell sensitivity to TRAIL.