Different mechanisms for CBFβ-MYH11 function in acute myeloid Leukemia (AML) with inv(16) have been proposed such as tethering of RUNX1 outside the nucleus, interference with transcription factor complex assembly and recruitment of histone deacetylases, all resulting in transcriptional repression of RUNX1 target genes. Here, through genome-wide CBFβ-MYH11 binding site analysis and quantitative interaction proteomics we found that CBFβ-MYH11 localizes to RUNX1 occupied promoters where it interacts with TAL1, FLI1 and TBP associated factors (TAFs) in the context of the hematopoietic transcription factors ERG, GATA2 and PU.1/SPI1 and the co regulators EP300 and HDAC1. Transcriptional analysis revealed that upon fusion protein knock down a small subset of the CBFβ-MYH11 target genes show increased expression, confirming a role in transcriptional repression. However, the majority of CBFβ-MYH11 target genes, including genes implicated in hematopoietic stem cell (HSC) self-renewal such as ID1, LMO1 and JAG1, are actively transcribed and repressed upon fusion protein knock down. Together these results suggest an essential role for CBFβ-MYH11 in regulating expression of genes involved in maintaining a stem cell phenotype.Leukemia advance online publication, 4 September 2013; doi:10.1038/leu.2013.257.