Abstract Bcl2 is associated with chemoresistance and poor prognosis in patients with various hematologic malignancies. DNA damage-induced p53/Bcl2 interaction at the outer mitochondrial membrane results in a Bcl2 conformational change with loss of its antiapoptotic activity in interleukin-3–dependent myeloid H7 cells. Here we find that specific disruption of protein phosphatase 2A (PP2A) activity by either expression of small t antigen or depletion of PP2A/C by RNA interference enhances Bcl2 phosphorylation and suppresses cisplatin-stimulated p53/Bcl2 binding in association with prolonged cell survival. By contrast, treatment of cells with C2-ceramide (a potent PP2A activator) or expression of the PP2A catalytic subunit (PP2A/C) inhibits Bcl2 phosphorylation, leading to increased p53/Bcl2 binding and apoptotic cell death. Mechanistically, PP2A-mediated dephosphorylation of Bcl2 in vitro promotes its direct interaction with p53 as well as a conformational change in Bcl2. PP2A directly interacts with the BH4 domain of Bcl2 as a docking site to potentially “bridge” PP2A to Bcl2's flexible loop domain containing the target serine 70 phosphorylation site. Thus, PP2A may provide a dual inhibitory effect on Bcl2's survival function by both dephosphorylating Bcl2 and enhancing p53-Bcl2 binding. Activating PP2A to dephosphorylate Bcl2 and/or increase Bcl2/p53 binding may represent an efficient and novel approach for treatment of hematologic malignancies.