Patients whose neuroblastoma tumors express high levels of brain-derived neurotrophic factor (BDNF) and TrkB have an unfavorable prognosis. Our previous studies indicated that BDNF activation of the TrkB signal transduction pathway blocked the cytotoxic effects of chemotherapeutic drugs via the phosphatidylinositol 3-kinase pathway. Akt is an important downstream target of phosphatidylinositol 3-kinase and functions to regulate cell survival, proliferation, and protein synthesis. In this study, we examined whether Akt is required and sufficient to mediate BDNF/TrkB protection of neuroblastoma cells from chemotherapy. Transient transfection of a constitutively active Akt (Akt-Myr) into TrkB-expressing SY5Y cells (TB8 cells) increases Akt activation and attenuates the cell death induced by chemotherapeutic reagents in the absence of BDNF. Furthermore, expression of a dominant-negative Akt (Akt-K179A) blocks the ability of BDNF to rescue TB8 cells from chemotherapy-induced cell death. Pharmacologic inhibition of Akt, with PIA6, a phosphatidylinositol ether lipid analogue (PIA), blocks BDNF-induced phosphorylation of Akt and the downstream target of Akt. PIA6 sensitizes neuroblastoma cells to chemotherapy and attenuates BDNF protection of neuroblastoma cells from chemotherapy-induced cell death. These results indicate that Akt is a key signaling component by which BDNF activation of the TrkB signal transduction pathway protects neuroblastoma cells from chemotherapy-induced cell death. This study raises the possibility that novel pharmacologic inhibitors of Akt may enhance the effectiveness of chemotherapeutic agents in the treatment of neuroblastoma tumors.