Vascular endothelial cells are continuously exposed to mechanical (e.g., shear stress) and chemical (e.g., growth factors) stimuli. It is important to elucidate the mechanisms by which cells perceive and integrate these different stimuli to regulate the downstream signaling pathways. We (50) have previously reported the shear-induced interplay between two membrane receptors, integrins and Flk-1. In the present study, we investigated the molecular mechanisms regulating the downstream IkappaB kinase (IKK) pathway in response to shear stress and VEGF. Both shear stress and VEGF induced a transient increase of IKK activity. These effects were inhibited by SU-1498, a specific Flk-1 inhibitor, and by a negative mutant of Casitas B-lineage lymphoma (Cbl) with tyrosine-to-phenylalanine mutations at sites 700, 731, and 774 (Cbl(nm)). Because Flk-1 and Cbl form a complex upon shearing or VEGF applications (50), these results suggest that shear stress and VEGF activate IKK via the receptor Flk-1 and its recruitment of the adapter protein Cbl. The inhibition of the shear- and VEGF-induced IKK activities by a negative mutant of Akt indicates that Akt acts upstream to IKK in response to shear stress and VEGF. Furthermore, SU-1498 and Cbl(-nm) abolished the shear- and VEGF-induced Akt activity, indicating that Akt acts at a level downstream to Flk-1 and Cbl. Therefore, our results indicate that the signaling events induced by shear stress and VEGF converge at the membrane receptor Flk-1 and that these stimuli share the Flk-1/Cbl/Akt pathway in activating IKK activation.