Objective-Smad4 is a central mediator of transforming growth factor-β/bone morphogenetic protein signaling that controls numerous developmental processes as well as homeostasis in the adult. The present studies sought to understand the function of Smad4 expressed in vascular smooth muscle cells (VSMC) in vascular development and the underlying mechanisms. Methods and Results-Breeding of Smad4 flox/flox mice with SM22α-Cre mice resulted in no viable offspring with SM22α-Cre;Smad4 flox/flox genotype in a total of 165 newborns. Subsequent characterization of 301 embryos between embryonic day (E) 9.5 and E14.5 demonstrated that mice with SM22α-Cre;Smad4 flox/flox genotype died between E12.5 and E14.5 because of decreased cell proliferation and increased apoptosis in the embryonic heart and arteries. Additionally, deletion of Smad4 more specifically in SMC with the inducible smooth muscle myosin heavy chain (SMMHC)-Cre mice, in which decreased cell proliferation was observed only in the artery but not the heart, also caused lethality of the knockout embryos at E12.5 and E14.5. The Smad4-deficient VSMC lacked smooth muscle α-actin filaments, decreased expression of SMC-specific gene markers, and markedly reduced cell proliferation, migration, and attachment. Using specific pharmacological inhibitors and small interfering RNAs, we demonstrated that inhibition of transforming growth factor-β signaling and its regulatory Smad 2/3 decreased VSMC proliferation, migration, and expression of SMC-specific gene markers, whereas inhibition of bone morphogenetic protein signaling only affected VSMC migration. Conclusion-SMC-specific deletion of Smad4 results in vascular defects that lead to embryonic lethality in mice, which may be attributed to decreased VSMC differentiation, proliferation, migration, as well as cell attachment and spreading. The transforming growth factor-β signaling pathway contributes to VSMC differentiation and function, whereas the bone morphogenetic protein signaling pathway regulates VSMC migration. These studies provide important insight into the role of Smad4 and its upstream Smads in regulating SMC function and vascular development of mice. © 2012 American Heart Association, Inc.