The interleukin-2 receptor β chain (IL-2Rβ) is expressed on a variety of hematopoietic cell types, including natural killer (NK) cells and nonconventional T lymphocyte subsets such as intestinal intraepithelial lymphocytes (IEL). However, the importance of IL-2Rβ-mediated signaling in the growth and development of these cells has yet to be clearly established. We have investigated IEL and NK cells in mice deficient for IL-2Rβ and describe here striking defects in the development of these cells. IL-2Rβ^−/− mice exhibited an abnormal IEL cell population, characterized by a dramatic reduction in T cell receptor αβ CD8αα and T cell receptor γδ lymphocytes. This selective decrease indicates that IEL can be classified into those whose development and/or differentiation is dependent on IL-2Rβ function and those for which IL-2Rβ–mediated signaling is not essential. NK cell development was also found to be disrupted in IL-2Rβ–deficient mice, characterized by a reduction in NK1.1⁺CD3⁻ cells in the peripheral circulation and an absence of NK cytotoxic activity in vitro. The dependence of NK cells and certain subclasses of IEL cells on IL-2Rβ expression points to an essential role for signaling through this receptor, presumably by IL-2 and/or IL-15, in the development of lymphocyte subsets of extrathymic origin.