Rho GTPase functions have been carefully investigated for many years using cell biological models. In recent years, mouse models with targeted mutations in Rho GTPase genes enabled the study of Rho GTPase function in vivo, partially confirming and partially contradicting expectations based on earlier in vitro experiments. This review sums up recent findings on the role of Rho GTPases in development, underlining the importance of in vivo research for our understanding of Rho GTPases in living organisms, and describing challenges for the future.