Fli-1 belongs to the Ets transcription factor family and is expressed in haematopoietic cells, including most of the cells that are active in immunity. The mononuclear phagocytes, i.e. monocytes, macrophages and dendritic cells, originate in haematopoietic stem cells and play an important role in immunity. To assess the role of Fli-1 in mononuclear phagocyte development in vivo, we generated mice that express a truncated Fli-1 protein, lacking the C-terminal transcriptional activation domain (Fli-1ΔCTA). Fli-1ΔCTA/ΔCTA mice had significantly increased populations of haematopoietic stem cells and common dendritic cell precursors in bone marrow compared with wild-type littermates. Significantly increased classical dendritic cells, plasmacytoid dendritic cells, and macrophage populations were found in spleens from Fli-1∆CTA/∆CTA mice compared with wild-type littermates. Fli-1ΔCTA/ΔCTA mice also had increased pre-classical dendritic cell and monocyte populations in peripheral blood mononuclear cells. Furthermore, bone marrow reconstitution studies demonstrated that expression of Fli-1 in both haematopoietic cells and stromal cells affected mononuclear phagocyte development in mice. Expression of Fms-like tyrosine kinase 3 ligand (Flt3L), a haematopoietic growth factor, in multipotent progenitors was statistically significantly increased from Fli-1∆CTA/∆CTA mice compared with wild-type littermates. Fli-1 protein binds directly to the promoter region of the Flt3L gene. Hence, Fli-1 plays an important role in the mononuclear phagocyte development, and the C-terminal transcriptional activation domain of Fli-1 negatively modulates mononuclear phagocyte development.