We have analyzed the immune system in Stat5-deficient mice. Although Stat5a-/- splenocytes have a partial defect in anti-CD3-induced proliferation that can be overcome by high dose interleukin (IL)-2, we now demonstrate that defective proliferation in Stat5b-/- splenocytes cannot be corrected by this treatment. Interestingly, this finding may be at least partially explained by diminished expression of the IL-2 receptor beta chain (IL-2Rbeta), which is a component of the receptors for both IL-2 and IL-15, although other defects may also exist. Similar to the defect in proliferation in activated splenocytes, freshly isolated splenocytes from Stat5b-/- mice exhibited greatly diminished proliferation in response to IL-2 and IL-15. This results from both a decrease in the number and responsiveness of natural killer (NK) cells. Corresponding to the diminished proliferation, basal as well as IL-2- and IL-15-mediated boosting of NK cytolytic activity was also greatly diminished. These data indicate an essential nonredundant role for Stat5b for potent NK cell-mediated proliferation and cytolytic activity.