Earlier studies have shown both p53-dependent and -independent tumor-suppressive functions of B56gamma-specific protein phosphatase 2A (B56gamma-PP2A). In the absence of p53, B56gamma-PP2A can inhibit cell proliferation and cell transformation by an unknown mechanism. In the presence of p53, on DNA damage, a complex including B56gamma-PP2A and p53 is formed, which leads to Thr55 dephosphorylation of p53, induction of the p53 transcriptional target p21 and inhibition of cell proliferation. In spite of its significance in inhibition of cell proliferation, no B56gamma mutations have been linked to human cancer to date. In this study, we first differentiate between the p53-dependent and -independent functions of B56gamma-PP2A by identifying a domain of the B56gamma protein required for interaction with p53. Within this region, we identify a B56gamma mutation, F395C, in lung cancer that disrupts the B56gamma-p53 interaction. More importantly, we show that F395C is unable to promote p53 Thr55 dephosphorylation, transcriptional activation of p21 and the p53-dependent tumor-suppressive function of PP2A. This finding provides a mechanistic basis for the p53-dependent and -independent functions of B56gamma-PP2A and establishes a critical link between B56gamma-PP2A p53-dependent tumor-suppressive function and tumorigenesis.