© 2013 Wiley Periodicals, Inc. Osteopontin (OPN), an adhesive, matricellular glycoprotein, is a rate-limiting factor in tumor promotion of skin carcinogenesis. With a tumor promotion model, the JB6 Cl41.5a cell line, we have shown that suppressing 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced OPN expression markedly inhibits TPA-induced colony formation in soft agar, an assay indicative of tumorigenic transformation. Further, the addition of exogenous OPN promotes colony formation of these cells. These findings support a function of OPN in mediating TPA-induced neoplastic transformation of JB6 cells. In regard to the mechanism of action by OPN, we hypothesized that, for JB6 cells grown in soft-agar, secreted OPN induced by TPA stimulates cell proliferation and/or prevents anoikis to facilitate TPA-induced colony formation. Analyses of cell cycle and cyclin D1 expression, and direct cell counting of JB6 cells treated with OPN indicate that OPN does not stimulate cell proliferation relative to non-treated controls. Instead, at 24h, OPN decreases anoikis by 41%, as assessed by annexin V assays. Further, in suspended cells OPN suppresses caspase-8 activation, which is mediated specifically through its RGD-cell binding motif that transduces signals through integrin receptors. Transfection studies with wild-type and mutant focal adhesion kinases (FAK) and Western blot analyses suggest that OPN suppression of caspase-8 activation is mediated through phosphorylation of FAK at Tyr861. In summary, these studies indicate that induced OPN is a microenvironment modulator that facilitates tumorigenic transformation of JB6 cells by inhibiting anoikis through its RGD-dependent suppression of caspase-8 activity, which is mediated in part through the activation of FAK at Tyr861.