Cells expressing the HIV-1 envelope glycoprotein complex (gp120/gp41, Env) induce the death of target cells either after cell-to-cell fusion or after cell-to-cell contact in a fusion-independent fashion. Here, we demonstrate that Env-induced death of single cells (including primary CD4 T cells) required gp120 and gp41 function. The gp41 peptide C34, which blocked syncytium formation, completely inhibited the death of single target cells by specifically acting on gp41 function. Moreover, Env-induced single cell death was exclusively observed in CD4 cells and was associated with specific gp41-mediated transfer of lipids from the membrane of Env-expressing cells to the target cell but not with detectable cytoplasm mixing (complete fusion). We conclude that after gp120 function, gp41 mediates close cell-to-cell contacts, thereby triggering cell death in single uninfected cells in the absence of detectable cell-to-cell fusion.