Abstractβ‐Catenin plays a dual role as a key effecter in the regulation of adherens junctions as well as a transcriptional co‐activator. Tyrosine phosphorylation of β‐catenin affects the cell adhesion, migration, and gene transcription in many types of human cancer cells, including acute myeloid leukemia cells with FLT3 internal tandem duplication (FLT3/ITD‐AML). Here, we investigated the relationship between three tyrosine residues (Y86, Y142, and Y654) in β‐catenin and oncogenic FLT3/ITD kinase. In the experiments using COS‐7 cells expressing FLT3/ITD and Wt or mutant β‐catenin, FLT3/ITD phosphorylated Y654, and this residue was essential for β‐catenin’s nuclear localization by FLT3/ITD. Promoter‐reporter assays demonstrated that Y654 phosphorylation of β‐catenin was closely related to TCF transcriptional activity. In vitro kinase assays, using recombinant FLT3 and biotinylated β‐catenin peptide including Y654 showed that FLT3 directly phosphorylated Y654 of β‐catenin. These results explain how FLT3/ITD affects the tyrosine phosphorylation, nuclear localization, and transcriptional activity of β‐catenin. Targeting Y654 phosphorylation may lead to the development of novel approaches to therapy for FLT3/ITD‐AML.