CD40 and CD40 ligand (CD40L) interaction is a key regulator of B-chronic lymphocytic leukemia (CLL) survival. CD40 activation leads to binding with tumor necrosis factor receptor-associated factors (TRAFs) and the subsequent activation of multiple downstream signaling pathways involved in cellular proliferation and survival. We have generated a novel fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, using XenoMouse® mice (Abgenix, Inc). CHIR-12.12 blocks CD40L binding to CD40 and inhibits CD40L-induced proliferation/survival of normal human B cells, primary CLL cells, and primary non-Hodgkin’s lymphoma (NHL) cells. We have also demonstrated that it has highly potent antibody-dependent cellular cytotoxicity (ADCC) against primary CLL and non-Hodgkin’s lymphoma cells. We have now investigated its effects on primary CLL cell survival. Soluble human CD40L prolongs primary CLL cell survival in culture, and treatment with CHIR-12.12 inhibits this survival when measured 48–72 hours after addition of CHIR-12.12. CD40L-mediated survival is associated with activation and phosphorylation of Akt, p38 MAPK, ERK, and IkB kinases a and b. Additionally, the anti-apoptotic proteins Mcl-1, Bcl-xl, and XIAP are induced, and markers of apoptosis (cleaved PARP and Caspase-3) are reduced. In contrast, CHIR-12.12 treatment of CD40L-stimulated primary CLL cells ex vivo inhibited downstream phosphorylation of Akt, p38 MAPK, ERK, and IkB kinases (IKK) a and b. Additionally, CHIR-12.12 treatment resulted in induction of cleaved caspase-3 and PARP, and reduction of XIAP, Mcl-1, and Bcl-xl expression, ultimately leading to CLL cell apoptosis. These results demonstrate that CHIR-12.12 inhibits CD40L-mediated signaling pathways and cell survival and could be a potential therapeutic treatment for CLL. CHIR-12.12 is currently in a Phase I clinical study for CLL.