Pathogen-associated molecular patterns (PAMPs), molecular moieties produced by invading microbial pathogens, initiate innate immune responses by binding to pattern recognition receptors (PRRs). Engagement of PRRs elicits a wide variety of responses, including the production and release of cytokines and chemokines. These responses require the activation of several parallel signaling pathways, including NF-kappaB, the interferon regulatory factors, and the MAPKs. The JNK and p38 MAPK groups are major PRR effectors and are key to the PRR-dependent induction and release of proinflammatory cytokines such as tumor necrosis factor and interleukin-8. The mammalian Ste20 orthologue germinal center kinase (GCK) is required for the activation of JNK by a subset of PAMPs; however, the mechanisms by which GCK couples to downstream events remain unclear. Here we show that GCK is required for JNK and, unexpectedly, p38 activation by three bacterial PAMPs, lipopolysaccharide, peptidoglycan, and flagellin (FliC). We show that these same PAMPs, in a GCK-dependent manner, activate mixed lineage kinases-2 and -3, MAPK kinase kinases upstream of JNK, and p38. We also show that MLK2 and -3 are required for activation of JNK and p38 by ectopically expressed GCK. Finally, we show that MLK2 and -3 are required for lipopolysaccharide, peptidoglycan, and FliC recruitment of JNK and p38 as well as for PAMP recruitment of the transcription factor c-Jun, and for the induction of interleukin-8. Our results define a signaling pathway whereby PAMPs can trigger MAPK activation and gene expression.