Cytokines and chemokines activate and direct effector cells during infection. We previously identified a functional group of five cytokines and chemokines, namely, IFN-γ, activation-induced T cell-derived and chemokine-related cytokine/lymphotactin, macrophage-inflammatory protein 1α, macrophage-inflammatory protein 1β, and RANTES, coexpressed in individual activated NK cells, CD8+ T cells, and CD4+ Th1 cells in vitro and during in vivo infections. However, the stimuli during infection were not known. In murine CMV (MCMV) infection, the DAP12/KARAP-associated Ly49H NK cell activation receptor is crucial for resistance through recognition of MCMV-encoded m157 but NK cells also undergo in vivo nonspecific responses to uncharacterized stimuli. In this study, we show that Ly49H ligation by m157 resulted in a coordinated release of all five cytokines/ chemokines from Ly49H+ NK cells. Whereas other cytokines also triggered the release of these cytokines/chemokines, stimulation was not confined to the Ly49H+ population. At the single-cell level, the production of the five mediators showed strong positive correlation with each other. Interestingly, NK cells were a major source of these five cytokines/chemokines in vitro and in vivo, whereas infected macrophages produced only limited amounts of macrophage-inflammatory protein 1α, macrophage-inflammatory protein1β, and RANTES. These findings suggest that both virus-specific and nonspecific NK cells play crucial roles in activating and directing other inflammatory cells during MCMV infection.