Maintenance of bone structural integrity depends in part on the rate of apoptosis of bone-forming osteoblasts. Since substrate adhesion is an important regulator of apoptosis, we investigated the role of focal adhesions in regulating bone cell apoptosis. To test this, we expressed a truncated form of -actinin (ROD-GFP) that competitively displaces endogenous -actinin from focal adhesions, thus disrupting focal adhesions. Immunofluorescence and morphometric analysis of vinculin and tyrosine phosphorylation revealed that ROD-GFP expression dramatically disrupted focal adhesion organization and reduced tyrosine phosphorylation at focal adhesions. Additionally, Bcl-2 protein levels were reduced in ROD-GFP-expressing cells, but neither caspase 3 cleavage, poly-ADP-ribose polymerase (PARP) cleavage, histone H2A.X phosphorylation, nor cytotoxicity were increased due to ROD-GFP expression alone. Increases in both ERK and Akt phosphorylation were also observed in ROD-GFP expressing cells, although inhibition of either ERK or Akt individually, or both together, failed to induce apoptosis. However, we did find that ROD-GFP expression sensitized, while -actinin-GFP expression protected, cells from TNF-induced apoptosis. Further investigation revealed that activation of TNF-induced survival signals, specifically Akt phosphorylation and NF-B activation, were inhibited in ROD-GFP-expressing cells. The reduced expression of anti-apoptotic Bcl-2 and inhibited survival signaling rendered ROD-GFP-expressing cells more susceptible to TNF-induced apoptosis. Thus, we conclude that -actinin plays a role in regulating cell survival through stabilization of focal adhesions and regulation of TNF- induced survival signaling.