The differentiation of haematopoietic cells is regulated by a plethora of so-called transcription factors (TFs). Mutations in genes encoding TFs or graded reduction in their expression levels can induce the development of various malignant diseases such as acute myeloid leukaemia (AML). Growth Factor Independence 1 (GFI1) is a transcriptional repressor with key roles in haematopoiesis, including regulating self-renewal of haematopoietic stem cells (HSCs) as well as myeloid and lymphoid differentiation. Analysis of AML patients and different AML mouse models with reduced GFI1 gene expression levels revealed a direct link between low GFI1 protein level and accelerated AML development and inferior prognosis. Here, we report that upregulated expression of GFI1 in several widely used leukemic cell lines inhibits their growth and decreases the ability to generate colonies in vitro. Similarly, elevated expression of GFI1 impedes the in vitro expansion of murine pre-leukemic cells. Using a humanized AML model, we demonstrate that upregulation of GFI1 expression leads to myeloid differentiation morphologically and immunophenotypically, increased level of apoptosis and reduction in number of cKit+ cells. These results suggest that increasing GFI1 level in leukemic cells with low GFI1 expression level could be a therapeutic approach.