Objectives: The Rho subfamily of small GTPases, including RhoA, Rac1, and Cdc42, regulates diverse cellular functions, including polarity and migration. Our prior studies established an essential role for Cdc42 in vascular network assembly, demonstrating that the genetic inactivation of Cdc42 yields defective vascular morphogenesis due to impaired migration of endothelial precursor cells. We have further shown that protein kinase Ciota and glycogen synthase kinase-3 Beta are downstream effectors of Cdc42 involved in mediating vascular network assembly. The objective of this study was to elucidate the guanine nucleotide exchange factors (GEFs) that activate Cdc42; specifically, we investigated the role of Zizimin1 and its effects on Cdc42 and vasculogenesis. Methods: We performed affinity pulldown assays using a nucleotide-free Cdc42 G15A mutant that specifically binds to Cdc42 GEFs. Mass spectrometric analysis identified Zizimin1 as a candidate Cdc42 GEF. Results: During vasculogenesis in embryoid bodies (EBs) differentiated from embryonic stem cells, Zizimin1 is highly expressed in aggregated endothelial cell precursors prior to vascular network assembly, mainly localizing to the Golgi apparatus. Surprisingly, stable overexpression of Zizimin1 in EBs resulted in inhibition of blood vessel formation, as evidenced by immunofluorescence microscopy demonstrating loss of vascular network development. Affinity pulldown assay showed that overexpression of Zizimin1 in fact increases Cdc42 activity; however, the activation of Rac1 and RhoA is significantly inhibited. Further, ectopically expressed Zizimin1 was also detected in the Golgi apparatus, co-localizing with Cdc42. Conclusions: Since Rac1 and RhoA signaling has been reported to play an essential role in blood vessel formation, our results suggest that the interplay between Rho GTPases guides vascular network assembly during development. Furthermore, we speculate that Zizimin1 overexpression, leading to overactivation of Cdc42 in the Golgi apparatus, may hinder cell migration. These findings provide novel insights into the mechanisms of embryonic vasculogenesis and also important new information for the design of potential pro- and/or anti- angiogenic therapies.