Hexamethylene bisacetamide (HMBA) is a potent inducer of cell differentiation and HIV production in chronically infected cells. However, its mechanism of action remains poorly defined. In this study, we demonstrate that HMBA activates transiently the PI3K/Akt pathway, which leads to the phosphorylation of HEXIM1 and the subsequent release of active positive transcription elongation factor b (P-TEFb) from its transcriptionally inactive complex with HEXIM1 and 7SK small nuclear RNA (snRNA). As a result, P-TEFb is recruited to the HIV promoter to stimulate transcription elongation and viral production. Despite the continuous presence of HMBA, the released P-TEFb reassembles rapidly with 7SK snRNA and HEXIM1. In contrast, a mutant HEXIM1 protein that cannot be phosphorylated and released from P-TEFb and 7SK snRNA via the PI3K/Akt pathway antagonizes this HMBA-mediated induction of viral production. Thus, our studies reveal how HIV transcription is induced by HMBA and suggest how modifications in the equilibrium between active and inactive P-TEFb could contribute to cell differentiation.