Aberrant activation of self-renewal pathways such as Wnt/β-catenin signalling pathway has been recognized underpinning malignant stem cells in different cancers. Although Wnt/β-catenin signalling has recently been shown to be required for the development of leukaemic stem cells (LSCs) in acute myeloid leukaemia (AML), its key pathway components remain largely unknown. Gene expression analysis of mixed lineage leukaemia (MLL)-rearranged AML patient samples revealed a 3-fold increase in expression of Lgr4, a receptor for the R-spondin (Rspo) family of Wnt agonists, in leukaemic cells compared to normal haematopoietic stem cells. Here we show that Lgr4 positively regulates β-catenin signalling during development of AML LSCs. Depletion of Lgr4 inhibited pre-LSC growth and significantly prolonged mouse survival. Chemical suppression of Gnaq attenuated Rspo/Wnt3a-Lgr4-mediated β-catenin transactivation, induced apoptosis and suppressed clonogenic growth of leukaemic cells. Furthermore, enforced expression of Lgr4 promoted cell proliferation in vivo and thereby accelerated the onset of AML via augmented activation of β-catenin. In conclusion, we report here an Rspo/Wnt3a-Lgr4-Gnaq-β-catenin signalling cascade in AML leukaemogenesis. Inhibition of the pathway components impairs leukaemia development, highlighting them as promising therapeutic targets for MLL AML.