Translocations involving the MLL gene at 11q23 are a frequent finding in therapy-related leukemia and are concentrated within a short, 8.3-kb tract of DNA, the breakpoint cluster region. In addition, a specific site adjacent to exon 12 within this region of MLL is cleaved in cells undergoing apoptosis. We show here, using human TK6 lymphoblastoid cells, that irradiation and the apoptotic trigger anti-CD95 antibody are each able to initiate translocations at the MLL exon 12 cleavage site. The translocation junctions produced contain regions of microhomology consistent with operation of the nonhomologous end joining (NHEJ) repair process. Participation of the NHEJ process is supported by the identification of the NHEJ component DNA-PKcs at the site of apoptotic cleavage. Suppression of DNA-PKcs function by the phosphatidylinositol 3-kinase inhibitor wortmannin compromises DNA end joining, increases site-specific cleavage within MLL, and eliminates MLL-restricted translocations. We propose that activation of apoptotic effector nucleases alone is sufficient to generate proleukemogenic translocations and raises the possibility that some of these may persist in cells that evade apoptotic execution and survive.