The oncofusion protein MLL-AF9 promotes acute myeloid leukemia (AML) by altering histone methylation and DNA accessibility to transcriptional machinery. Using antagonists of WNT signaling and genetic ablation of the WNT chaperone Wntless (WLS), we identified the homeobox domain gene sine oculis homeobox homolog 1 (Six1) to be a WNT-controlled target gene in MLL-AF9-transformed leukemic initiating cells (LICs). Wls or Six1 disruption in MLL-AF9 LICs, or exposure of these cells to WNT pathway inhibitors results in extended survival in LIC-transplanted recipient mice suggesting that WNT/SIX1 signaling is critical to MLL-AF9 transformation. Profiling of chromatin accessibility changes in MLL-AF9 LICs using ATAC-seq reveals chromatin remodeling in Six1 DNA regions that support binding of the transcriptional effector TCF7L2. Finally we show human AML cell lines with MLL-rearrangements exhibit elevated Six1 expression and are sensitive to a WNT inhibitor in a Six1-dependent fashion. Our data suggests de novo installation of a WNT/SIX1 signaling axis is critical to the development of some forms of AML and reveals how epigenetic alterations can rewire cell fate regulatory pathways with cancer-promoting consequences. Funding Statement: This work was supported by the Welch Foundation (I-1665; LL), CPRIT (RP130212; LL and CC), the National Cancer Institute (1R01 CA168761; JK), and P50-CA70907 Declaration of Interests: L. Lum, J. Lu, H. Shi, C. Chen, and L.S. Zhang are named inventors on patents covering various WNT inhibitors including those targeting PORCN. Ethics Approval Statement: All animal experiments were performed with the approval of the Institutional Animal Care & Use Committee (IACUC) at the University of Texas Southwestern Medical Center (UTSWMC).