FMS-like tyrosine kinase 3 (FLT3) normally functions in the survival/proliferation of hematopoietic stem/progenitor cells, but its constitutive activation by internal tandem duplication (ITD) mutations correlates with a poor prognosis in AML. The development of FLT3 tyrosine kinase inhibitors (TKI) is a promising strategy, but resistance that arises during the course of treatment caused by secondary mutations within the mutated gene itself poses a significant challenge. In an effort to predict FLT3 resistance mutations that might develop in patients, we used saturation mutagenesis of FLT3/ITD followed by selection of transfected cells in FLT3 TKI. We identified F621L, A627P, F691L and Y842C mutations in FLT3/ITD that confer varying levels of resistance to FLT3 TKI. Western blotting confirmed that some FLT3 TKI were ineffective at inhibiting FLT3 autophosphorylation and signaling through MAP kinase, STAT5 and AKT in some mutants. Balb/c mice transplanted with the FLT3/ITD Y842C mutation confirmed resistance to sorafenib in vivo but not to lestaurtinib. These results indicate a growing number of FLT3 mutations that are likely to be encountered in patients. Such knowledge, combined with known remaining sensitivity to other FLT3 TKI, will be important to establish as secondary drug treatments that can be substituted when these mutants are encountered.