C57BL/6 mice genetically deficient in interleukin 15 (IL-15^−/− mice) were generated by gene targeting. IL-15^−/− mice displayed marked reductions in numbers of thymic and peripheral natural killer (NK) T cells, memory phenotype CD8⁺ T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes (IELs). The reduction but not absence of these populations in IL-15^−/− mice likely reflects an important role for IL-15 for expansion and/or survival of these cells. IL-15^−/− mice lacked NK cells, as assessed by both immunophenotyping and functional criteria, indicating an obligate role for IL-15 in the development and functional maturation of NK cells. Specific defects associated with IL-15 deficiency were reversed by in vivo administration of exogenous IL-15. Despite their immunological defects, IL-15^−/− mice remained healthy when maintained under specific pathogen-free conditions. However, IL-15^−/− mice are likely to have compromised host defense responses to various pathogens, as they were unable to mount a protective response to challenge with vaccinia virus. These data reveal critical roles for IL-15 in the development of specific lymphoid lineages. Moreover, the ability to rescue lymphoid defects in IL-15^−/− mice by IL-15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine.