The TLR family of pattern recognition receptors is largely responsible for meditating the activation of macrophages by pathogens. Because macrophages may encounter multiple TLR ligands during an infection, signaling crosstalk between TLR pathways is likely to be important for the tailoring of inflammatory reactions to pathogens. Here, we show that rather than inducing tolerance, LPS pretreatment primed the inflammatory response (e.g., TNF production) of mouse bone marrow-derived macrophages (BMM) to the TLR9 ligand, CpG DNA. The priming effects of LPS, which correlated with enhanced Erk1/2, JNK, and p38 MAPK activation, appeared to be mediated via both c-Fms-dependent and -independent mechanisms. LPS pretreatment and inhibition of the M-CSF receptor, c-Fms, with GW2580 had comparable effects on CpG DNA-induced Erk1/2 and p38 MAPK activation. However, c-Fms inhibition did not enhance CpG DNA-induced JNK activation; also, the levels of TNF produced were significantly lower than those from LPS-primed BMM. Thus, the priming effects of LPS on TLR9 responses appear to be largely mediated via the c-Fms-independent potentiation of JNK activity. Indeed, inhibition of JNK abrogated the enhanced production of TNF by LPS-pretreated BMM. The c-Fms-dependent priming effects of LPS are unlikely to be a consequence of the inhibitory constraints of M-CSF signaling on TLR9 expression being relieved by LPS; instead, LPS may exert its priming effects via signaling molecules downstream of TLR9. In summary, our findings highlight the importance of signaling crosstalk between TLRs, as well as between TLRs and c-Fms, in regulating the inflammatory reaction to pathogens.