Mutations of the gene encoding the FLT3 tyrosine kinase are the most common mutation in AML. Recently Wnt-dependent signaling has been suggested to cooperate with FLT3 in myeloid transformation. In the work presented here we have examined the mechanism of the interaction of FLT3 and the Wnt effector β-catenin in FLT3 leukemia cells. In comparison to cells with wild-type FLT3, cells with activating mutations of FLT3 including internal tandem duplication (ITD) of the juxtamembrane domain of FLT3 and the FLT3 D835Y mutation had elevated levels of tyrosine-phosphorylated β-catenin. Although β-catenin was located mainly in the cytoplasm in WT-FLT3 cell lines, it was primarily nuclear in cells with FLT-3 activating mutations. Treatment of activated FLT3 cells with the FLT3 kinase inhibitor AG1296 decreased tyrosine-phoshorylation of β-catenin and downregulated the level of nuclear β-catenin. Treatment of WT-FLT3 cell lines with FLT3 ligand increased tyrosine-phosphorylation of β-catenin and upregulated its nuclear accumulation. Immunoprecipitation experiments demonstrated that FLT3-ITD binds to β-catenin and treatment with AG1296 decreased interaction between FLT3-ITD and β-catenin. In vitro kinase assays using recombinant FLT3 and β-catenin showed that an active FLT3 kinase phosphorylates tyrosine residues of β-catenin directly and AG1296 decreased the activated FLT3-induced tyrosine-phosphorylation of β-catenin. The data demonstrate that activated FLT3 induces tyrosine phosphorylation of β-catenin and accumulation of nuclear β-catenin. We propose that increased β-catenin nuclear signaling is a potential mechanism of activated FLT-3-associated malignant transformation.