Chronic myeloid leukemia (CML) is the most common subtype of chronic myeloproliferative diseases. The disease typically progresses through three clinicopathologic phases: chronic (CP), accelerated (AP), and blast (BP). CML is characterized by the Philadelphia chromosome, which involves the t(9;22)(q34;q11). This aberrant translocation generates the Bcr-Abl fusion gene that leads to constitutive activation of Abl kinase. Abl appears to play a major role in the pathogenesis of CML. In this regard, Abl was shown to interact with a wide array of molecules directly involved in several important cellular biologic functions. Targeted inhibition of Abl, such as by imatinib, has provided an effective therapeutic approach in CML. However, a subset of CML patients, particularly those in BP, demonstrates resistance to imatinib. Janus kinase 3 (Jak3) is a protein tyrosine kinase that plays a role in regulating cell survival primarily by phosphorylation/activation of signal transducers and activators of transcription (Stats). The role of Jak3 in CML is not known. Using Western blotting and immunohistochemical staining, we showed that Jak3 and its activated/phosphorylated form (pJak3) are expressed in two CML cell lines K562 and KBM-5. To study the biologic effects of Jak3 inhibition in CML, we utilized two selective inhibitors of Jak3: WHI-P131 and WHI-P154 (Calbiochem, San Diego, CA), which induced concentration-dependent decrease in pJak3, but not in Jak3. The decrease in pJak3 was associated with a gradual decrease in pStat3 and pStat5. These changes lead to several biologic alterations including decreased cell viability and proliferation, apoptotic cell death, and cell cycle arrest. These alterations were due to significant decrease in Bcl-XL, Mcl-1, and cyclin D3, and upregulation of p27. To explore possible functional interaction between Jak3 and Abl, we performed co-immunoprecipitation studies that demonstrated Jak3 and Abl to be physically associated. Importantly, using an in vitro kinase assay, selective inhibition of Jak3 decreased Abl kinase activity to 61% of its baseline level at 6 h after treatment, which suggests that Jak3 contributes to the constitutive activation of Abl. In further support of the biologic importance of Jak3 in CML, we examined the frequency of expression of Jak3 and pJak3 in 34 bone marrow specimens collected from 25 CML patients classified based on the WHO classification scheme (CP: 9 [7 F; median age = 41 y], AP: 8 [2 F; 56.5 y], BP: 8 [1 F; 53 y]). There was a gradual increase in cells expressing Jak3 with the progression of the disease (48.5%±9.9 in CP, 65.0%±9.6 in AP, and 84.4%±4.0 in BP; P < 0.01 for CP vs. BP, Kruskal-Wallis test). Notably, in the same study cases, pJak3 showed pronounced expression in BP compared with CP and AP (12.0%±6.8 in CP, 15.7%±7.5 in AP, and 70.0%±7.5 in BP; P < 0.001 for CP vs. BP and P < 0.01 for AP vs. BP, Kruskal-Wallis test). In conclusion, our data provide novel evidence that Jak3 plays a significant role in the pathogenesis of CML, at least in part via activation of Abl. In addition, Jak3 expression and activation become more pronounced with the progression of CML from chronic to blast phase. These findings identify Jak3 as a potential therapeutic target in CML, particularly in blast phase where patients are more likely to be resistant to imatinib.