Abstract Lack of target selectivity is a well-known limitation to the use of chemical inhibitors to pinpoint individual kinase functions in the intricate network of cellular signaling pathways that often involve closely related kinases or unrelated off-target effects. A possible solution to this problem involves the use of inhibitor-resistant mutants of the targeted kinase. Such mutants can be employed in rescue experiments, in which introduction of the mutant kinase reverses the phenotype resulting from chemical inhibition of the endogenous kinase. This chemical genetics approach has been successfully used by our group to dissect roles for individual members of the Src kinase family in embryonic stem cell differentiation and chronic myelogenous leukemia, despite close structural homology within this kinase family. In the present study, we used this approach to validate the specificity of a series of potent inhibitors of the c-Fes kinase that we recently identified in a kinase-biased chemical library screen. The c-Fes protein-tyrosine kinase is involved in multiple cellular signaling pathways that modulate differentiation, the innate immune response, vasculogenesis and tumorigenesis. Using site-directed mutagenesis, we systematically altered residues around the inhibitor-binding pocket defined in a recent crystal structure of the c-Fes SH2-kinase region bound to the diaminopyrimidine inhibitor, TAE684. We first mutated the gatekeeper methionine (Met636) of Fes, which helps mediate TAE684 binding in the crystal, but found that substitutions at this position inactivated kinase function. Additional point mutations that retained kinase activity were then tested against TAE684 and other potent small molecule inhibitors of c-Fes. This screen identified a leucine residue near the gatekeeper position (Leu638) to be critical in conferring sensitivity to both TAE864 and the more Fes-selective pyrazolopyrimidine, WZ-4-49-8. Replacement of this Leu638 with a bulkier tyrosine or phenylalanine rendered c-Fes very resistant to WZ-4-49-8, with IC50 values increasing from the low nanomolar range for wild-type Fes to the micromolar range for the mutants in an in vitro kinase assay. These data implicate Leu638 as an important specificity determinant for future design of more potent and selective inhibitors of c-Fes. Ongoing studies are using this resistant mutant to validate novel roles for Fes in biological systems impacted by TAE684 and WZ-4-49-8 treatment, including differentiation of macrophages (a major site of c-Fes expression) to osteoclasts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A234.