Chronic lymphocytic leukemia (CLL) is characterized by clonal accumulation of CD5(+) CD19(+) B lymphocytes that are arrested in the G(0)/G(1) phase of the cell cycle and fail to undergo apoptosis because of overexpression of the antiapoptotic B-cell CLL/lymphoma 2 (BCL-2) protein. Oncolytic viruses, such as vesicular stomatitis virus (VSV), have emerged as potential anticancer agents that selectively target and kill malignant cells via the intrinsic mitochondrial pathway. Although primary CLL cells are largely resistant to VSV oncolysis, we postulated that targeting the apoptotic pathway via inhibition of BCL-2 may sensitize CLL cells to VSV oncolysis. In the present study, we examined the capacity of EM20-25--a small-molecule antagonist of the BCL-2 protein--to overcome CLL resistance to VSV oncolysis. We demonstrate a synergistic effect of the two agents in primary ex vivo CLL cells (combination index of 0.5; P < 0.0001). In a direct comparison of peripheral blood mononuclear cells from healthy volunteers with primary CLL, the two agents combined showed a therapeutic index of 19-fold; furthermore, the combination of VSV and EM20-25 increased apoptotic cell death in Karpas-422 and Granta-519 B-lymphoma cell lines (P < 0.005) via the intrinsic mitochondrial pathway. Mechanistically, EM20-25 blocked the ability of the BCL-2 protein to dimerize with proapoptotic BAX protein, thus sensitizing CLL to VSV oncolytic stress. Together, these data indicate that the use of BCL-2 inhibitors may improve VSV oncolysis in treatment-resistant hematological malignancies, such as CLL, with characterized defects in the apoptotic response.