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Background: Patients with relapsed/refractory acute myeloid leukaemia (AML) with FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) have limited treatment options and poor prognosis. Therefore, novel treatment modalities are needed. Since high expression of natural killer group 2 member D ligands (NKG2DLs) can be induced by FLT3 inhibitors, we constructed dual-target FLT3 single-chain fragment variable (scFv)/NKG2D-chimeric antigen receptor (CAR) T cells, and explored whether FLT3 inhibitors combined with FLT3scFv/NKG2D-CAR T cells could have synergistic anti-leukaemia effects. Methods: FLT3scFv and NKG2D expression in CAR T cells, FLT3 and NKG2DL expression in AML cells, and the in vitro cytotoxicity of combining CAR T cells with gilteritinib were assessed by flow cytometry. The therapeutic effect was evaluated in a xenograft mouse model established by injection of MOLM-13 cells. Mechanisms underlying the gilteritinib-induced NKG2DL upregulation were investigated using siRNA, ChIP-QPCR and luciferase assays. Results: The FLT3scFv/NKG2D-CAR T cells specifically lysed AML cells both in vitro and in the xenograft mouse model. The efficacy of FLT3scFv/NKG2D-CAR T cells was improved by gilteritinib-pretreatment. The noncanonical NF-κB2/Rel B signalling pathway was found to mediate gilteritinib-induced NKG2DL upregulation in AML cells. Conclusions: Bispecific FLT3scFv/NKG2D-CAR T cells can effectively eradicate AML cells. The FLT3 inhibitor gilteritinib can synergistically improve this effect by upregulating NF-κB2-dependent NKG2DL expression in AML cells. [ABSTRACT FROM AUTHOR]