p53 not only functions as a transcription factor but also has a direct extranuclear apoptogenic role at the mitochondria. We have discovered that DNA damage-induced p53/Bcl2 binding is associated with decreased Bcl2/Bax interaction and increased apoptotic cell death in a mechanism regulated by Bcl2’s flexible loop regulatory domain (FLD) in IL3-dependent myeloid NSF/N1.H7 cells. Purified p53 protein can disrupt the Bcl2/Bax complex by directly binding to a negative regulatory region of the FLD (aa32–68). Deletion of the negative regulatory region (aa32–68) abolishes Bcl2/ p53 binding and enhances its antiapoptotic function. Removal of aa69–87 of the FLD, which contains Bcl2’s phosphorylation site(s) (T69, S70 and S87), enhances Bcl2/p53 binding and abrogates Bcl2’s potent survival activity, indicating this is a positive regulatory region. Phosphorylation of Bcl2 in the FLD inhibits p53 binding since the phosphomimetic T69E/S70E/S87E (EEE) but not the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutant displays a reduced capacity to bind p53 and more potently inhibits p53-induced cytochrome c release. A full-length Bcl2 loop-only protein (aa32–87) can directly bind p53 to impede Bcl2/p53 binding in vitro. Either DNA damage or expression of p53 targeted to mitochondria results in Bcl2/p53 binding followed by exposure of Bcl2’s BH3 domain and inactivation of Bcl2’s antiapoptotic function. Therefore, Bcl2’s FLD contains both a positive and negative regulatory regions which functionally regulate Bcl2’s antiapoptotic activity via differential binding of Bax and p53, respectively.