Mutation or epigenetic silencing of the transcription factor C/EBPα is observed in ∼10% of patients with acute myeloid leukemia (AML). In both cases, a common global gene expression profile is observed, but downstream targets relevant for leukemogenesis are not known. Here, we identify Sox4 as a direct target of C/EBPα whereby its expression is inversely correlated with C/EBPα activity. Downregulation of Sox4 abrogated increased self-renewal of leukemic cells and restored their differentiation. Gene expression profiles of leukemia-initiating cells (LICs) from both Sox4 overexpression and murine C/EBPα mutant AML models clustered together but differed from other types of AML. Our data demonstrate that Sox4 overexpression resulting from C/EBPα inactivation contributes to the development of leukemia with a distinct LIC phenotype. •C/EBPα represses Sox4 transcription in a DNA-binding-dependent manner•SOX4 is overexpressed in human AML samples with mutated or silent CEBPA•Sox4 mediates leukemic outgrowth due to defective C/EBPα in murine and human models•LICs in Sox4- or mutated C/EBPα-driven AML models share a gene expression signature