The DNA-end-joining reactions used for repair of double-strand breaks in DNA and for V(D)J recombination, the process by which immunoglobulin and T-cell antigen-receptor genes are assembled from multiple gene segments, use common factors. These factors include components of DNA-dependent protein kinase (DNA-PK), namely DNA-PKcs and the Ku heterodimer, Ku70-Ku80, and XRCC4. The precise function of XRCC4 is unknown, but it interacts with DNA ligase IV. Ligase IV is one of the three known mammalian DNA ligases; however, the in vivo functions of these ligases have not been determined unequivocally. Here we show that inactivation of the ligase IV gene in mice leads to late embryonic lethality. Lymphopoiesis in these mice is blocked and V(D)J joining does not occur. Ligase IV-deficient embryonic fibroblasts also show marked sensitivity to ionizing radiation, growth defects and premature senescence. All of these phenotypic characteristics, except embryonic lethality, resemble those associated with Ku70 and Ku80 deficiencies, indicating that they may result from an impaired end-joining process that involves both Ku subunits and ligase IV. However, Ku-deficient mice are viable, so ligase IV must also be required for processes and/or in cell types in which Ku is dispensable.