Dysregulated cytokine responsiveness by myeloid cells can be a trigger for the development of chronic inflammation as well as inflammatory bowel disease. Thus, mice with a myeloid-specific defect in signal transducer and activator of transcription (Stat) 3 develop spontaneous colitis secondary to the inability of myeloid cells to respond to the immunosuppressive cytokine interleukin-10. We now examined whether the inflammation caused by Stat3-deficient macrophages is cell autonomous or dependent on their interaction with lymphocytes. For this purpose, myeloid-specific Stat3-deficient mice (LysMcre/Stat3(flox) mice) were intercrossed with RAG-1 knockout mice to generate LysMcre/Stat3(flox) RAG(-/-) mice. In these mutants and LysMcre/Stat3(flox) littermate control mice we determined the onset and severity of spontaneous chronic enterocolitis, and the reaction to dextran sodium sulphate (DSS)-induced epithelial damage, as well as to lipopolysaccharide (LPS) challenge. In contrast to LysMcre/Stat3(flox) mice, LysMcre/Stat3(flox) RAG(-/-) animals are protected from chronic enterocolitis. Although they respond to oral dextran sulphate with acute colitis symptoms, the inflammation heals similarly to wild type mice whereas LysMcre/Stat3(flox) mice exhibit continued colitis pathology. In addition, the hyperreactivity of LysMcre/Stat3(flox) mice to LPS-challenge in vivo was less severe in the absence of lymphocytes. Despite clear differences in the strength of inflammatory responses, macrophages of both LysMcre/Stat3(flox) mice and LysMcre/Stat3(flox) RAG(-/-) animals exhibited increased costimulatory capacity. In conclusion, our findings demonstrate that Stat3-deficient myeloid cells alone are not capable of inducing the severe pathology seen in LysMcre/Stat3(flox) mice. Yet when these cells can interact with lymphocytes their increased costimulatory potential will trigger an overshooting inflammatory response.