Aberrant activation of beta-catenin is a common event in AML and is an independent predictor of poor prognosis. Although increased beta-catenin signaling in AML has been associated with oncogenic translocation products and activating mutations in the FLT3R, the mechanisms that activate beta-catenin in AML more broadly are still unclear. Here, we describe a novel link between IL-3 signaling and the regulation of beta-catenin in myeloid transformation and AML. In a murine model of HoxB8 and IL-3 cooperation, we show that beta-catenin protein levels are modulated by IL-3 and that Cre-induced deletion of beta-catenin abolishes IL-3-dependent growth and colony formation. In IL-3-dependent leukemic TF-1.8 cells, we observed increased beta-catenin protein levels and nuclear localization in response to IL-3, and this correlated with transcriptional induction of beta-catenin target genes. Furthermore, IL-3 promoted beta-catenin accumulation in a subset of AML patient samples, and gene-expression profiling of these cells revealed induction of WNT/beta-catenin and TCF4 gene signatures in an IL-3-dependent manner. This study is the first to link beta-catenin activation to IL-3 and suggests that targeting IL-3 signaling may be an effective approach for the inhibition of beta-catenin activity in some patients with AML.