Fli1 is a member of the Ets family of transcription factors and is preferentially expressed in hematopoietic cell lineages. Its expression level is linked to the pathogenesis of lupus. In this study, we identified mechanisms involved in the transcriptional regulation of the mouse and human Fli1 promoters. We show that the Fli1 promoter is upregulated by Ets factors Ets1, Ets2, Fli1 and Elf1 either alone or in combination with GATA factors, but is inhibited by Tel. In vitro binding studies show that Elf1, Tel and Fli1 in T cells bind the three Ets-binding sites in the murine Fli1 proximal promoter. We identified transcription factor-binding sites in the human Fli1 promoter region that function in T cells in a similar manner to those in the mouse promoter. Furthermore, we show similar binding of Ets factors to the endogenous mouse and human Fli1 promoters in T cells and knocking down Ets1 results in an upregulation of Fli1 expression. Together, these results suggest that the human and mouse genes are regulated similarly and that Ets1 may be important in preventing the overexpression of Fli1 in T cells. This report lays the groundwork for identifying targets for manipulating Fli1 expression as a possible therapeutic approach.