Summary: DNA double-strand breaks (DSBs) represent dangerous chromosomal lesions that can lead to mutation, neoplastic transformation, or cell death. DSBs can occur by extrinsic insult from environmental sources or may occur intrinsically as a result of cellular metabolism or a genetic program. Mammalian cells possess potent and efficient mechanisms to repair DSBs, and thus complete normal development as well as mitigate oncogenic potential and prevent cell death. When DSB repair (DSBR) fails, chromosomal instability results and can be associated with tumor formation or progression. Studies of mice deficient in various components of the non-homologous end joining pathway of DSBR have revealed key roles in both the developmental program of B and T lymphocytes as well as in the maintenance of general genome stability. Here, we review the current thinking about DSBs and DSBR in chromosomal instability and tumorigenesis, and we highlight the implications for understanding the karyotypic features associated with human tumors.