During development, T and B cells encountering their cognate ligands via antigen-specific receptors are deleted or rendered anergic. Like T and B cells, natural killer (NK) cells express certain receptors, such as Ly49H, associated with immunoreceptor tyrosine-based activation motif-bearing adaptor proteins that transmit activating signals through Syk family kinases. Ly49H binds with high affinity to a mouse cytomegalovirus (MCMV)-encoded glycoprotein, m157, but does not recognize self-antigens. For comparison with the behavior of immature T and B cells exposed to foreign antigens, we addressed the fate of Ly49H(+) NK cells that encountered their viral ligand during development by retroviral transduction of bone marrow stem cells with m157. In chimeric mice expressing m157, we observed a reduction in Ly49H(+) NK cells in multiple tissues and less Ly49H on the cell surface. NK cells exposed to m157 during development appeared less mature, produced less interferon gamma when stimulated through Ly49H, and were unable to kill m157-bearing target cells. After MCMV infection, these NK cells were severely impaired in their ability to proliferate. Thus, if immature NK cells encounter ligands for their activating receptors, regulatory mechanisms exist to keep these cells in an unresponsive state.