Significance Plexins, a family of transmembrane receptors for semaphorins, control diverse biological processes during mouse development. However, it is largely unknown through which signaling pathways they exert their functions in vivo. Using an allelic series of transgenic mice, we show that the GTPase activating protein domain of plexins constitutes their key signaling module during development, which is required for proper formation of the nervous, cardiovascular, and skeletal system. In contrast, development of the liver vasculature specifically depends on the activation of the small GTPase RhoA by the plexin family member Plexin-B2. This study uncovers the in vivo context-dependence and functional specificity of individual plexin-mediated signaling pathways during mouse development.