A hallmark of mixed lineage leukemia gene-rearranged (MLL-r) acute myeloid leukemia (AML) that offers an opportunity for targeted therapy is addiction to protein-tyrosine kinase (TK) signaling. One such signaling is the receptor TK (RTK) Fms-like receptor tyrosine kinase 3 (FLT3) upregulated by cooperation of the transcription factors homeobox A9 (HOXA9) and Meis homeobox 1 (MEIS1). Signal peptide-CUB-EGF-like repeat-containing protein (SCUBE) family previously shown to act as a co-receptor for augmenting signaling activity of an RTK (e.g., vascular endothelial growth factor receptor). However, whether SCUBE1 is involved in the pathological activation of FLT3 during MLL-r leukemogenesis remains unknown. Here we first show that SCUBE1 is a direct target of HOXA9/MEIS1 that is highly expressed on the MLL-r cell surface and predicts poor prognosis in de novo AML. We further demonstrate by using a conditional knockout mouse model that Scube1 is required for both the initiation and maintenance of MLL-AF9–induced leukemogenesis in vivo. Further proteomic, molecular and biochemical analyses reveal that the membrane-tethered SCUBE1 binds to the FLT3 ligand and the extracellular ligand-binding domains of FLT3, thus facilitating activation of the signal axis FLT3-LYN (a nonreceptor TK) to initiate leukemic growth and survival signals. Importantly, targeting surface SCUBE1 by an anti-SCUBE1 monomethyl auristatin E antibody–drug conjugate led to significantly decreased cell viability specifically in MLL-r leukemia. Our study reports a novel function of SCUBE1 in leukemia and unravels the molecular mechanism of SCUBE1 in MLL-r AML. Thus, SCUBE1 is a potential therapeutic target for treating leukemia caused by MLL rearrangements.