Patients with chronic lymphocytic leukemia (CLL) treated with adenovirus (Ad)-CD154 (CD40L) gene therapy experience reductions in leukemia cell counts and lymph node size associated with induction of the death receptor Fas (CD95). CD4 T cell lines can induce apoptosis of CD40-activated CLL cells via a CD95 ligand (CD95-L)-dependent mechanism. To examine whether CD95-L was sufficient to induce cytolysis of CD40-activated CLL cells, we used Chinese hamster ovary cells transfected with CD95-L as cytotoxic effector cells. CD40-activated CLL cells were initially resistant to CD95-mediated apoptosis despite high-level expression of CD95. However, after 72 h, CLL cells from seven of seven patients became increasingly sensitive to CD95-mediated apoptosis. This sensitivity correlated with a progressive decline in Flice-inhibitory protein (FLIP), which was induced within 24 h of CD40 ligation. Down-regulation of FLIP with an antisense oligonucleotide or a pharmacologic agent, however, was not sufficient to render CLL cells sensitive to CD95-mediated apoptosis in the 24–72 h after CD40 activation. Although the levels of pro-Caspase-8 appeared sufficient, inadequate levels of Fas-associated death domain protein (FADD) and DAP3 may preclude assembly of the death-inducing signaling complex. Seventy-two hours after CD40 ligation, sensitivity to CD95 and a progressive increase in FADD and DAP3 were associated with the acquired ability of FADD and FLIP to coimmunoprecipitate with the death-inducing signaling complex after CD95 ligation. Collectively, these studies reveal that CD40 ligation on CLL B cells induces a programmed series of events in which the cells initially are protected and then sensitized to CD95-mediated apoptosis through shifts in the balance of the anti- and proapoptotic proteins FLIP and FADD.