Insertional mutagenesis resulting from the integration of retroviral vectors has led to the discovery of many oncogenes associated with leukemia. We investigated the role of HOXC6, identified by proximal provirus integration in a large animal hematopoietic stem cell gene therapy study, for a potential involvement in hematopoietic stem cell activity and hematopoietic cell fate decision. HOXC6 was overexpressed in the murine bone marrow transplantation model and tested in a competitive repopulation assay in comparison to the known hematopoietic stem cell expansion factor, HOXB4. We have identified HOXC6 as a factor that enhances competitive repopulation capacity in vivo and colony formation in vitro. Ectopic HOXC6 expression also induced strong myeloid differentiation and expansion of granulocyte-macrophage progenitors/common myeloid progenitors (GMPs/CMPs) in vivo, resulting in myeloid malignancies with low penetrance (3 of 17 mice), likely in collaboration with Meis1 because of a provirus integration mapped to the 3' region in the malignant clone. We characterized the molecular basis of HOXC6-induced myeloid differentiation and malignant cell transformation with complementary DNA microarray analysis. Overexpression of HOXC6 induced a gene expression signature similar to several acute myeloid leukemia subtypes when compared with normal GMPs/CMPs. These results demonstrate that HOXC6 acts as a regulator in hematopoiesis and is involved in malignant transformation.