Inhibition of the JAK2/STAT pathway has been recently implicated in cytoprotective mechanisms in both vascular smooth muscle cells and astrocytes. The advent of JAK2 specific inhibitors provides a practical tool for the study of this pathway in different cellular types. The interest of finding methods to improve endothelial cell (EC) resistance to injury led us to examine the effect of JAK2/STAT inhibition on EC protection. Furthermore, the signaling pathways involved in JAK2/STAT inhibition-related actions were examined. Our results reveal, for the first time, that blockade of JAK2 with the tyrosine kinase inhibitor, AG490, strongly protects cultured EC against cell detachment-dependent anoikis and serum deprivation, and increases reseeding efficiency. Confirmation of the specificity of the effects of JAK2 inhibition was attained by finding protective effects upon transfection with a dominant negative JAK2. Furthermore, AG490 blocked serum deprivation-induced phosphorylation of JAK2. In terms of mechanism, treatment with AG490 induces several relevant responses, both in monolayer and detached cells. These mechanisms include: 1. Increase and nuclear translocation of the active, dephosphorylated -catenin. This translocation was transcriptionally active and its protective effect was further supported by the stimulation of EC cytoprotection by transfectionally-induced excess of -catenin. 2. Increase of PECAM/CD31 levels. 3. Increase in total and phosphorylated AKT. 4. Increase in phosphorylated GSK3/. The present findings imply potential practical applications of JAK2 inhibition on EC. These applications affect not only EC in monolayer, but also circulating, detached cells, and involve previously undescribed mechanistic interactions.