We report that the erythropoietin receptor cytosolic juxtamembrane region is conformationally rigid and contains a hydrophobic motif, composed of residues L²⁵³, I²⁵⁷, and W²⁵⁸, that is crucial for Janus kinase 2 (JAK2) activation and receptor signaling. Alanine insertion mutagenesis shows that the orientation of this motif and not its distance from the membrane bilayer is critical. Intragenic complementation studies suggest that L²⁵³ is contained within an α helix functionally continuous to the transmembrane α helix. The α-helical orientation of L²⁵³ is required not for JAK2 activation but for activated JAK2 to induce phosphorylation of the erythropoietin receptor. This motif is highly conserved among cytokine receptors and couples ligand-induced conformational changes in the receptor to intracellular activation of JAK2.