Evi‐1 is a zinc‐finger transcriptional factor whose inappropriate expression leads to leukemic transformation in mice and humans. Recently, it has been shown that Evi‐1 regulates proliferation of hematopoietic stem/progenitor cells at embryonic stage via GATA‐2 up‐regulation; however, detailed mechanisms underlying Evi‐1‐mediated early hematopoiesis are not fully understood. We therefore evaluated hematopoietic potential of Evi‐1 mutants using a cultivation system of murine para‐aortic splanchnopleural (P‐Sp) regions, and found that both the first zinc finger domain and the acidic domain were required for Evi‐1‐mediated hematopoiesis. The hematopoietic potential of Evi‐1 mutants was likely to be related to its ability to up‐regulate GATA‐2 expression. We also showed that the decreased colony forming capacity of Evi‐1‐deficient P‐Sp cells was successfully recovered by inhibition of TGF‐b signaling, using ALK5 inhibitor or retroviral transfer of dominant‐negative‐type Smad3. Our findings suggest that Evi‐1 promotes hematopoietic stem/progenitor expansion at the embryonic stage through up‐regulation of GATA‐2 and repression of TGF‐b signaling. (Cancer Sci 2008; 99: 1407–1413)