The fludarabine and cyclophosphamide couplet has become the backbone of the chronic lymphocytic leukemia (CLL) standard of care. Although this is an effective treatment, it results in untoward toxicity. Bendamustine is a newly approved and better-tolerated alkylating agent. We hypothesized that similar to cyclophosphamide, bendamustine-induced DNA damage will be inhibited by fludarabine, resulting in increased cytotoxicity. To test this hypothesis and the role of the stromal microenvironment in this process, we treated CLL lymphocytes in vitro with each drug alone and in combination. Simultaneous or prior addition of fludarabine to bendamustine resulted in maximum cytotoxicity assayed by 3,3'-dihexyloxacarbocyanine iodine negativity, annexin positivity, and poly (adenosine 5'-diphosphate-ribose) polymerase cleavage. Cytotoxicity elicited by combination of both agents was similar in these malignant B cells cultured either in suspension or on marrow stroma cells. Cell death was associated with DNA damage response, which was determined by phosphorylation of H2AX and unscheduled DNA synthesis. H2AX activation was maximum with the drug combination, and unscheduled DNA synthesis induced by bendamustine was blocked by fludarabine. In parallel, ATM, Chk2, and p53 were phosphorylated and PUMA was induced. Cell death was caspase independent; however, caspases did decrease levels of Mcl-1 survival protein. These data provide a rationale for combining fludarabine with bendamustine for patients with CLL.