Interleukin-10 (IL-10) is intensely studied, yet little is known about the mechanisms that control IL-10 expression. We identified striking similarities between IL-10 and interferon-γ (IFN-γ) regulation in mouse natural killer (NK) cells. Like IFN-γ, IL-10 expression is induced by IL-2 and IL-12 and IL-2 + IL-12 stimulation is synergistic. Unlike IFN-γ, neither IL-18 nor Ly-49D cross-linking induced IL-10 expression however. Additionally, the IL-12 homologs IL-23 and IL-27 also do not regulate NK cell-specific IL-10. We determined that a small population of NK cells accounts for IL-10 production. The induction of IL-10 by IL-2 + IL-12 treatment in NK cells appears to be biphasic, with an initial burst of expression which diminishes by 12 h but spikes again at 18 h. We determined that much like IFN-γ, Stat4 is largely required for IL-12-induced IL-10. Conversely, we observed normal induction of IL-10 in T-bet-deficient NK cells. We identified a Stat4-binding element in the fourth intron of the Il10 gene, which is completely conserved between mouse and human. This intronic Stat4 motif is within a conserved noncoding sequence, which is also a target for cytokine-induced histone acetylation. These findings highlight tissue- and receptor-specific IL-10 regulatory mechanisms, which may be part of an early feedback loop.