We hypothesize that this relapse may be due mutations in the JAK2 kinase domain that prevent inhibitor binding, as is the case with IM-treated BCRABL. Using a random mutagenesis approach, we have identified JAK2 kinase domain residues critical in evading small-molecule inhibition. Here we describe the identification and characterization of mutations in the JAK2 kinase domain that confer resistance to the presence of small-molecule inhibitors in vitro. Inhibitor resistance is currently one of the biggest challenges facing Doxorubicin Topoisomerase inhibitor effective treatment of CML. Evidence Staurosporine suggests that BCRABL mutations are present at the commencement of treatment, and the inhibitor provides strong selective pressure for affected clone outgrowth and consequent patient relapse. Considerable effort has been put forth in identifying and testing new generations of inhibitors targeting specific BCR-ABL mutations. The in vitro prediction of BCR-ABL mutations against multiple inhibitors was robust and provided the field with significant data to aid in the design of second and third generation kinase inhibitors. Identification of a single point mutation, JAK2 V617F, thought to play an important role in MPN development and progression, initiated the search for small-molecule inhibitors of the JAK2 tyrosine kinase. We hypothesized that inhibitor-resistant JAK2 alleles may become apparent as large cohorts of MPN patients progress through clinical trials testing JAK2-selective drug therapies. The objective of our study was to identify JAK2 mutations that provide resistance to small molecule inhibitors before patient relapse is observed in the clinic. Some variation in the activation of Stat5, Akt and Erk1/2 was observed in the absence of inhibitors with the inhibitor-resistant mutants. TEL-JAK2 mutants with elevated basal phosphorylation of downstream signaling components correlated with lower in vitro kinase activity. For example, TELJAK2 V881A had high Erk2 phosphorylation in the absence of JAK Inhibitor-I, but weak kinase activity upon drug addition. We also examined growth ability in the presence of two clinically relevant inhibitors, TG101348 and CEP-701. The lack of growth difference observed in the XTT data suggests we have isolated compound-specific, not ATP competitor-specific, mutations. To further understand how the JAK2 kinase domain has been modified by the presence of mutations, we developed a novel intracellular assay to directly assess its phosphorylation ability in a system more relevant than a standard in vitro kinase assay.