AbstractJAK2 activation is crucial for cytokine receptor signal transduction and leukemogenesis. However, the underlying processes that lead to full activation of JAK2 are unclear. Here, we report a positive role for ubiquitination of JAK2 during GM-CSF-induced activation. Upon GM-CSF stimulation, JAK2 ubiquitination is significantly enhanced through K63-linked poly-ubiquitination. Studies employing both knockout and overexpression of Cbl, an E3 ubiquitin ligase, led to the conclusion that Cbl specifically promotes JAK2 ubiquitination, and this was further confirmed in vitro using a Cbl ubiquitination assay. Moreover, following GM-CSF stimulation, the levels of phospho-JAK2 and -STAT5 and a STAT5 luciferase reporter assay were all reduced in Cbl knockout cells and this effect could be rescued by Cbl expression. Mechanistically, Cbl can interact with, and ubiquitinate JAK2 FERM and kinase domains via the Cbl TKB domain. Using lysine-to-arginine site-directed mutagenesis, K970 in the kinase domain of JAK2 was identified as the ubiquitination site important for promoting full JAK2 activation by Cbl via K63-conjugated poly-ubiquitination. Our study suggests that GM-CSF-induced JAK2 activation is enhanced by Cbl-mediated ubiquitination of JAK2. Targeting ubiquitination of JAK2 might offer a novel therapeutic strategy against JAK2-mediated disorders.