Jak family tyrosine kinases mediate signaling by cytokine receptors to regulate diverse biological processes. Although Jak2 and other Jak kinase family members are phosphorylated on numerous sites during cytokine signaling, the identity and function of most of these sites remains unknown. Using tandem mass spectroscopic analysis of activated Jak2 protein from intact cells, we identified Tyr₂₂₁ and Tyr₅₇₀ as novel sites of Jak2 phosphorylation. Phosphorylation of both sites was stimulated by cytokine treatment of cultured cells, and this stimulation required Jak2 kinase activity. While we observed no gross alteration of signaling upon mutation of Tyr₂₂₁, Tyr₅₇₀ lies within the inhibitory JH2 domain of Jak2, and mutation of this site (Jak2^Y570F) results in constitutive Jak2-dependent signaling in the absence of cytokine stimulation and enhances and prolongs Jak2 activation during cytokine stimulation. Mutation of Tyr₅₇₀ does not alter the ability of SOCS3 to bind or inhibit Jak2, however. Thus, the phosphorylation of Tyr₅₇₀ in vivo inhibits Jak2-dependent signaling independently of SOCS3-mediated inhibition. This Tyr₅₇₀-dependent mechanism of Jak2 inhibition likely represents an important mechanism by which cytokine function is regulated.