Gene targeting and overexpression studies have demonstrated the importance of the clustered homeobox (HOX) genes in hematopoesis. In addition, global HOX gene dysregulation is found in the majority of cases of acute myeloid leukemia (AML) and many cases of acute lymphoblastic leukemia (ALL), and substantial evidence exists to suggest that aberrant expression of HOX genes contributes to the pathogenesis of leukemia. However, although individual HOX genes are rearranged in rare cases of AML and HOX genes are known transcriptional targets of certain leukemia-associated fusion proteins, such as those involving the mixed lineage leukemia (MLL) gene, in the majority of cases, the upstream regulators of HOX genes are unknown. The CDX family of non-clustered homeobox genes are known developmental regulators of HOX gene expression. We have recently demonstrated that Cdx4 is expressed in adult murine bone marrow where its expression pattern follows that of Hox genes. We also demonstrated that CDX2 is expressed in the majority, and that CDX4 is expressed in almost a quarter, of AML patient samples. For CDX2, this expression was predominantly monoallelic but was not associated with coding sequence or promoter mutations, gene amplification, or aberrant promoter methylation. In addition, stable knockdown of CDX2 resulted in a loss of proliferation and clonogenicity in AML cell lines, and bone marrow retrovirally engineered to express either Cdx2 or Cdx4 generated AML in transplant recipients. Cdx4 was shown to cooperate with the known Hox cofactor Meis1a, and structure-function experiments confirmed that the transcription factor function of Cdx4 was required for transformation. Finally, expression of either Cdx2 or Cdx4 generated a dysregulated Hox gene program in normal hematopoietic progenitors and in leukemic tissue. Taken together, these studies implicate CDX proteins as master regulators of HOX gene regulation in AML.