Negative regulation of cytokine signaling is critical for the generation of the appropriate cellular outcome in response to signals, and can be modulated by other concomitant extracellular stimuli ("crosstalk"). Using both genetic and pharmacological manipulations we have investigated the mechanisms by which the pro-inflammatory stimuli, lipopolysaccharide (LPS) and Tumor necrosis factor alpha (TNFalpha), negatively regulate interleukin-6 (IL-6) signaling in primary mouse macrophages. Analysis of suppressor of cytokine signalling 3 (SOCS3)-deficient macrophages reveal that SOCS3 is necessary but surprisingly, not sufficient for the complete crosstalk inhibition of IL-6 signaling induced by LPS and TNFalpha. Analysis of macrophages from gp130 (Y757F) mutant mice suggest that SH2 domain-containing tyrosine phosphatase (SHP2) activity does not explain the residual inhibitory effect of these pro-inflammatory stimuli. In addition, p38 mitogen-activated protein kinase (p38) activation also negatively regulates IL-6 signaling independent of its parallel and necessary action to induce SOCS3 expression. Finally, we have identified an additional, novel mechanism of crosstalk inhibition: a reduction in total cellular levels of gp130 following stimulation with LPS and TNFalpha.