Focal adhesion kinase (FAK) is important to cellular functions such as proliferation, migration, and survival of anchorage-dependent cells. We investigated the role of FAK in modulating normal cellular responses, specifically cell survival in response to inflammatory stimuli and serum withdrawal, using FAK-knockout (FAK^-/-) embryonic fibroblasts. FAK^-/- fibroblasts were more vulnerable to TNF- induced apoptosis, as measured by terminal deoxynucleotidyl transferase (TdT)-positivity. FAK^-/- fibroblasts also demonstrated increased procaspase-3 cleavage to p17 subunit, whereas this was undetectable in FAK^+/+ fibroblasts. Insulin receptor substrate-1 (IRS-1) expression was completely abolished and nuclear factor-B (NF-B) activity was reduced, with a concomitant decrease in abundance of the anti-apoptotic protein Bcl-xL in FAK ^-/- cells. Upon serum withdrawal, FAK^+/+ cells exhibited marked attenuation of basal ERK phosphorylation, while FAK^-/- cells, in contrast, maintained high basal ERK phosphorylation. Moreover, inhibition of ERK phosphorylation potentiated serum withdrawal-induced caspase-3 activity. This was paralleled by increased IRS-2 expression in FAK^-/- cells, although both insulin- and IGF-1-mediated phosphorylation of Akt/PKB and GSK-3 were impaired. This suggests that IRS-2 protects against apoptosis upon serum-withdrawal via the ERK signaling pathway. The specific role of FAK to protect cells from apoptosis is regulated by activation and phosphorylation of NF-B and interaction between activated growth factor anti-apoptotic signaling pathways involving both PI3-K/Akt and MAPK/ERK1/2. We demonstrate that FAK is necessary for up-regulation of the anti-apoptotic NF-B response, as well as for normal expression of growth factor signaling proteins. Thus, we propose a novel role for FAK in protection from cytokine-mediated apoptosis.