Endothelial cells (EC) translate biomechanical forces into functional and phenotypic responses that play important roles in cardiac development. Specifically, EC in areas of high shear stress, i.e., in the cardiac outflow tract and atrioventricular canal, are characterized by high expression of Krüppel-like factor 2 (Klf2) and by transforming growth factor-beta (Tgfβ)-driven endothelial-to-mesenchymal transition. Extraembryonic venous obstruction (venous clip model) results in congenital heart malformations, and venous clip-induced alterations in shear stress-related gene expression are suggestive for an increase in cardiac shear stress. Here, we study the effects of shear stress on Klf2 expression and Tgfβ-associated signaling in embryonic EC in vivo using the venous clip model and in vitro by subjecting cultured EC to fluid flow. Cellular responses were assessed by analysis of Klf2, Tgfβ ligands, and their downstream signaling targets. Results show that, in embryonic EC, shear stress activates Tgfβ/Alk5 signaling and that induction of Klf2 is an Alk5 dependent process. Developmental Dynamics 240:1670–1680, 2011. © 2011 Wiley-Liss, Inc.