Interleukin-10 (IL-10) is an anti-inflammatory cytokine that modulates innate and adaptive immunity. IL-10 transcripts and the protein were induced in murine bone marrow-derived dendritic cells (BMDCs) after toll-like receptor (TLR) stimulation. IL-10 induction was TLR ligand selective, in that CpG DNA, imidazoquinolin, peptidoglycan, and zymosan but not lipopolysaccharide (LPS) and poly I:C led to IL-10 production. IL-10 induction was, however, completely absent in MyD88(/) DCs that lacked a TLR adaptor showing that IL-10 induction depends on TLR signaling. Kinetic analysis of IL-10 induction by CpG and imidazoquinolin revealed a prolonged lag phase prior to a measurable rise in transcript levels, which peaked at 12-24 h after stimulation. Stat3, implicated in IL-10 gene transcription, was also induced after TLR stimulation with the kinetics similar to those of IL-10 induction. Further, Stat3 was phosphorylated and bound to the IL-10 promoter in TLR-stimulated DCs. Supporting a link with IL-10 induction, STAT3 induction was absent in MyD88(/) DCs. These data suggest a two-step model where the initial TLR signaling induced proinflammatory cytokines, which then activated Stat3, leading to the induction of IL-10. TLR-stimulated IL-10 production may regulate DC maturation steps, thereby influencing the ensuing immune responses.