Flt1, an "fms-like tyrosine kinase" receptor, has been suggested to play an active role in vascular endothelial growth factor (VEGF)-mediated autocrine signaling of tumor growth and angiogenesis. Here, we used a neuroblastoma model to investigate the role of VEGF/Flt1 signaling in hypoxia-mediated tumor cell survival, drug resistance, and in vivo angiogenesis. SK-N-BE2, a highly malignant neuroblastoma cell line resistant to hypoxia-induced apoptosis expresses active Flt1 but lacks VEGFR2 expression. We found that 24-hour hypoxia (<0.1% O2) alone (no serum deprivation) showed sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) associated with bcl-2 up-regulation and resistance to etoposide-induced (5 mumol/L) apoptosis. Treatment with anti-VEGF and anti-Flt1 antibodies inhibited ERK1/2 activation, down-regulated bcl-2, and reversed the hypoxia-mediated drug resistance to etoposide. Similar results were obtained with U0126 and ursolic acid, specific and nonspecific inhibitors of ERK1/2, respectively. We confirmed the protective role of Flt1 receptor by small interfering RNA knockout and Flt1 overexpression studies. Subsequently, we found that inhibition of VEGF/Flt1 autocrine signaling led to reduced hypoxia-inducible factor-1alpha (HIF-1alpha) phosphorylation. Furthermore, the reduced phosphorylation was associated with down-regulation of basic fibroblast growth factor, a downstream target of the HIF-1alpha and VEGF pathways. Our findings suggested an expanded autocrine loop between VEGF/Flt1 signaling and HIF-1alpha. We investigated the angiogenic activity of the loop in an in vivo Matrigel plug assay. The hypoxia-treated conditioned medium induced a strong angiogenic response, as well as the cooption of surrounding vessels into the plugs; ursolic acid inhibited the angiogenesis process. We also found that three other Flt1-expressing neuroblastoma cell lines show hypoxia-mediated drug resistance to etoposide, melphalan, doxorubicin, and cyclophosphamide. Taken together, we conclude that a hypoxia-driven VEGF/Flt1 autocrine loop interacts with HIF-1alpha through a mitogen-activated protein kinase/ERK1/2 pathway in neuroblastoma. The interaction, in the form of an autocrine loop, is required for the hypoxia-driven cell survival, drug resistance, and angiogenesis in neuroblastoma.