Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that promotes cell migration, survival, and gene expression. Here we show that FAK signaling is important for tumor necrosis factor-α (TNFα)-induced interleukin 6 (IL-6) mRNA and protein expression in breast (4T1), lung (A549), prostate (PC-3), and neural (NB-8) tumor cells by FAK short hairpin RNA knockdown and by comparisons of FAK-null (FAK-/-) and FAK +/+ mouse embryo fibroblasts. FAK promoted TNFα-stimulated MAPK activation needed for maximal IL-6 production. FAK was not required for TNFα-mediated nuclear factor-κB or c-Jun N-terminal kinase activation. TNFα-stimulated FAK catalytic activation and IL-6 production were inhibited by FAK N-terminal but not FAK C-terminal domain overexpression. Analysis of FAK-/- fibroblasts stably reconstituted with wild type or various FAK point mutants showed that FAK catalytic activity, Tyr-397 phosphorylation, and the Pro-712/713 proline-rich region of FAK were required for TNFα-stimulated MAPK activation and IL-6 production. Constitutively activated MAPK kinase-1 (MEK1) expression in FAK-/- and A549 FAK short hairpin RNA-expressing cells rescued TNFα-stimulated IL-6 production. Inhibition of Src protein-tyrosine kinase activity or mutation of Src phosphorylation sites on FAK (Tyr-861 or Tyr-925) did not affect TNFα-stimulated IL-6 expression. Moreover, analyses of Src-/-, Yes-/-, and Fyn-/- fibroblasts showed that Src expression was inhibitory to TNFα-stimulated IL-6 production. These studies provide evidence for a novel Src-independent FAK to MAPK signaling pathway regulating IL-6 expression with potential importance to inflammation and tumor progression. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.