Objectives: The Rho subfamily of small GTPases, including RhoA, Rac1, and Cdc42, regulates diverse cellular functions, including polarity, migration, and actin-based cytoskeleton dynamics. 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-3b are downstream effectors of Cdc42 and are involved in mediating vascular network assembly. However, the guanine nucleotide exchange factors (GEFs) that activate Cdc42, remain unknown. Methods: We performed affinity pulldown assays using a nucleotide-free Cdc42 G15A mutant that specifically binds to Cdc42 GEFs. Mass spectrometric analysis identified Zizimin1, an upstream regulatory protein, 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 before vascular network formation. Surprisingly, stable overexpression of Zizimin1 in EBs resulted in the inhibition of blood vessel formation compared with control, evidenced by immunohistochemistry demonstrating loss of vascular network development. Affinity pulldown assay helped to elucidate that overexpression of Zizimin1 increases Cdc42 activity; however, the activation of Rac1 and RhoA is significantly inhibited. Conclusions: Because Rac1 and RhoA signaling has been reported to play an essential role in embryonic blood vessel formation, our results suggest that the interplay betweenRhoGTPases guides vascular network assembly during development. Furthermore, these findings provide novel insights into the mechanisms of embryonic vasculogenesis and also important new information for the design of potential proangiogenic and/or antiangiogenic therapies.