Vascular endothelial cells are continuously exposed to mechanical and chemical stimuli, such as shear stress and VEGF, respectively. It is still not clear how cells perceive these stimuli and orchestrate their responses. Studying the molecular mechanism by which shear stress and VEGF regulate the signaling pathways in bovine endothelial aortic cells, we found that VEGF induced a rapid association of VEGF receptor 2 (Flk-1) with Nck beta, but shear stress did not have such an effect. SU1498 (a specific inhibitor of Flk-1) and Nck beta(nm) (a negative mutant of Nck beta) blocked the VEGF-induced ERK and JNK activities. Only SU1498, but not Nck beta(nm), inhibited the shear-induced ERK activity. Furthermore, neither SU1498 nor Nck beta(nm) had significant effects on the shear-induced JNK activity, which can be blocked by inhibitors of Src family kinase and ROCK kinase. Therefore, mechanical (shear stress) and chemical (VEGF) stimuli diverge at the receptor Flk-1 in terms of the recruitment of the adapter protein Nck beta, and they employ different components of the complex signaling network in regulating downstream molecules, e.g., ERK and JNK.