Mutations in Fms-like tyrosine kinase 3 (FLT3) have been implicated in the pathogenesis of acute myeloid leukemia (AML) by affecting the proliferation and differentiation of hematopoietic stem and progenitor cells. Although several FLT3 inhibitors have been developed, the occurrence of secondary TKD mutations of FLT3 such FLT3/D835Y and FLT3/F691L lead to drug resistance and has become a key area of unmet medical needs. To overcome the obstacle of secondary TKD mutations, a new series of indirubin-3'-aminooxy-acetamide derivatives was discovered as potent and selective FLT3 and FLT3/D835Y inhibitors that were predicted to bind at the DFG-in active conformation of FLT3 in molecular docking studies. Through structure-activity relationship studies, the most optimized compound 13a was developed as a potent inhibitor at FLT3 and FLT3/D835Y with IC50 values of 0.26 nM and 0.18 nM, respectively, which also displayed remarkably strong in vitro anticancer activities, with single-digit nanomolar GI50 values for several AML (MV4-11 and MOLM14) and Ba/F3 cell lines expressed with secondary TKD mutated FLT3 kinases as well as FLT3-ITD. The selectivity profiles of compound 13a in the oncology kinase panel and various human cancer cell lines were prominent, demonstrating that its inhibitory activities were mainly focused on a few members of the receptor tyrosine kinase family and AML versus solid tumor cell lines. Furthermore, significant in vivo anticancer efficacy of compound 13a was confirmed in a xenograft animal model implanted with FLT3-ITD/D835Y-expressing MOLM-14 cells related to secondary TKD mutation.