IL-15 is a cytokine playing a crucial role in the function of immune cells, including NK and CD8 T cells. In this study, we demonstrated that in vivo, in mice, IL-15–prestimulated NK cells were no longer able to respond to a second cycle of IL-15 stimulation. This was illustrated by defects in cell maturation, proliferation, and activation, seemingly linked to the environment surrounding NK cells but not related to the presence of CD4 regulatory T cells, TGF-β, or IL-10. Moreover, NK cells from immunodeficient mice could respond to two cycles of IL-15 stimulation, whereas an adoptive transfer of CD44 + CD8 + cells impaired their responsiveness to the second cycle. Conversely, in immunocompetent mice, NK cell responsiveness to a second IL-15 stimulation was restored by the depletion of CD8 + cells. These biological findings refine our understanding of the complex mode of action of NK cells in vivo, and they should be taken into consideration for IL-15–based therapy.