Elucidating signaling pathways that mediate cell survival or apoptosis will facilitate the development of targeted therapies in cancer. In neuroblastoma tumors, brain-derived neurotrophic factor (BDNF) and its receptor TrkB are associated with poor prognosis. Our previous studies have shown that BDNF activation of TrkB induces resistance to chemotherapy via activation of phosphoinositide-3-kinase (PI3K)/Akt pathway. To study targets of PI3K/Akt that mediate protection from chemotherapy, we focused on glycogen synthase kinase-3beta (GSK-3beta), which is a known modulator of apoptosis. We used pharmacologic and genetic methods to study the role of GSK-3beta in the BDNF/TrkB/PI3K/Akt protection of neuroblastoma from chemotherapy. BDNF activation of TrkB induced the Akt-dependent phosphorylation of GSK-3beta, resulting in its inactivation. Treatment of neuroblastoma cells with inhibitors of GSK-3beta, LiCl, GSK-3beta inhibitor VII, kenpaullone, or a GSK-3beta-targeted small interfering RNA (siRNA) resulted in a 15% to 40% increase in neuroblastoma cell survival after cytotoxic treatment. Transfection of neuroblastoma cells with a constitutively active GSK-3beta S9A9 caused a 10% to 15% decrease in cell survival. Using real-time, dynamic measurements of cell survival, we found that 6 to 8 h after etoposide treatment was the period during which critical events regulating the induction of cell death or BDNF/TrkB-induced protection occurred. During this period, etoposide treatment was associated with the dephosphorylation and activation of GSK-3beta in the mitochondria that was blocked by BDNF activation of TrkB. These data indicate that the inactivation of GSK-3beta contributes to the BDNF/TrkB/PI3K/Akt protection of neuroblastoma cells from chemotherapy.