Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis and a high degree of relapse seen in patients. Overexpression of FMS-like tyrosine kinase 3 (FLT3) is associated with up to 70% of AML patients. Wild-type FLT3 induces proliferation and inhibits apoptosis in AML cells, while uncontrolled proliferation of FLT3 kinase activity is also associated with FLT3 mutations. Therefore, inhibiting FLT3 activity is a promising AML therapy. Flavonoids are a group of phytochemicals that can target protein kinases, suggesting their potential antitumor activities. In this study, several plant-derived flavonoids have been identified with FLT3 inhibitory activity. Among these compounds, compound 40 (5,7,4'-trihydroxy-6-methoxyflavone) exhibited the most potent inhibition against not only FLT3 (IC50 = 0.44 μM) but also FLT3-D835Y and FLT3-ITD mutants (IC50 = 0.23 and 0.39 μM, respectively). The critical interactions between the FLT3 binding site and the compounds were identified by performing a structure-activity relationship analysis. Furthermore, the results of cellular assays revealed that compounds 28, 31, 32, and 40 exhibited significant cytotoxicity against two human AML cell lines (MOLM-13 and MV-4-11), and compounds 31, 32, and 40 resulted in cell apoptosis and G0/G1 cell cycle arrest. Collectively, these flavonoids have the potential to be further optimized as FLT3 inhibitors and provide valuable chemical information for the development of new AML drugs.