Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) that mediate antiviral and antitumor responses and require the transcriptional regulator Eomesodermin (Eomes) for early development. However, the role of Eomes and its molecular program in mature NK cell biology is unclear. To address this, we develop a tamoxifen-inducible, type-1-ILC-specific (Ncr1-targeted) cre mouse and combine this with Eomes-floxed mice. Eomes deletion after normal NK cell ontogeny results in a rapid loss of NK cells (but not ILC1s), with a particularly profound effect on penultimately mature stage III NK cells. Mechanisms responsible for stage III reduction include increased apoptosis and impaired maturation from stage II precursors. Induced Eomes deletion also decreases NK cell cytotoxicity and abrogates in vivo rejection of major histocompatibility complex (MHC)-class-I-deficient cells. However, other NK cell functional responses, and stage IV NK cells, are largely preserved. These data indicate that mature NK cells have distinct Eomes-dependent and -independent stages. Display omitted •Induced Eomes deletion results in a rapid decrease in NK cell numbers•Eomes-deleted stage III NK cells exhibit increased apoptosis•Eomes-deleted stage II and III NK cells exhibit differentiation defects•Induced Eomes deletion compromises NK cytotoxicity and MHCI−/− rejection in vivo The transcription factor Eomes is important for early natural killer (NK) cell development. Wagner et al. utilize an inducible, type 1 ILC-specific cre model to demonstrate a stage-specific role for Eomes in NK cell survival and homeostasis as well as a persistent requirement for Eomes in promoting NK cell cytotoxicity.