The EVI1 gene (3q26) codes for a transcription factor with important roles in normal hematopoiesis and leukemogenesis. High expression of EVI1 is a negative prognostic indicator of survival in acute myeloid leukemia (AML) irrespective of the presence of 3q26 rearrangements. However, the only known mechanisms that lead to EVI1 overexpression are 3q aberrations, and the MLL-ENL oncoprotein, which activates the transcription of EVI1 in hematopoietic stem cells. Our aim was to characterize the functional promoter region of EVI1, and to identify transcription factors involved in the regulation of this gene. Generation of seven truncated constructs and luciferase reporter assays allowed us to determine a 318-bp region as the minimal promoter region of EVI1. Site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays identified RUNX1 and ELK1 as putative transcription factors of EVI1. Furthermore, knockdown of RUNX1 and ELK1 led to EVI1 downregulation, and their overexpression to upregulation of EVI1. Interestingly, in a series of patient samples with AML at diagnosis, we found a significant positive correlation between EVI1 and RUNX1 at protein level. Moreover, we identified one of the roles of RUNX1 in the activation of EVI1 during megakaryocytic differentiation. EVI1 knockdown significantly inhibited the expression of megakaryocytic markers after treating K562 cells with TPA, as happens when knocking down RUNX1. In conclusion, we define the minimal promoter region of EVI1 and demonstrate that RUNX1 and ELK1, two proteins with essential functions in hematopoiesis, regulate EVI1 in AML. Furthermore, our results show that one of the mechanisms by which RUNX1 regulates the transcription of EVI1 is by acetylation of the histone H3 on its promoter region. This study opens new directions to further understand the mechanisms of EVI1 overexpressing leukemias.Oncogene advance online publication, 11 June 2012; doi:10.1038/onc.2012.222.