Trans-membrane (or T cell) immunoglobulin and mucin (TIM) molecules are known regulators of immune response whose function in hematopoiesis is unknown. Earlier, we found that tim-1 and tim-4 are expressed by CD45(+) cells in the para-aortic region of chicken embryo. Because the para-aortic region is a known site for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) differentiation and expansion, we hypothesize that TIM molecules have a role in hematopoiesis. To study this role further, we analyzed TIM expression more precisely in chicken para-aortic region and mouse fetal liver hematopoietic cells. Additionally, we examined the hematopoietic potential of TIM-4(+) mouse fetal liver cells with a colony-forming assay. tim-1 gene expression was detected in chicken and mouse embryos in the aorta-gonads-mesonephros-region at the time of HSC emergence, whereas tim-3 mRNA was widely expressed in different tissues. tim-4 expression was restricted to fetal liver CD45(+)F4/80(+) cells. Moreover, two TIM-4(+) populations were distinguished: F4/80(hi)TIM-4(hi) and F4/80(lo)TIM-4(lo). F4/80(hi)TIM-4(hi) cells had no hematopoietic potential and were morphologically similar to mature macrophages, suggesting that they are yolk sac-derived macrophages. Instead, many of the F4/80(lo)TIM-4(lo) cells were c-kit(+) and Sca-1(+) and had primitive morphology and multilineage colony-forming ability. In addition, F4/80(lo)TIM-4(lo) cells included a considerable population expressing ER-MP12, a known marker for macrophage colony-forming cells and other myeloid progenitors. We conclude that TIM molecules are expressed in embryonic hematopoietic tissues in chicken and mouse and that in fetal liver, TIM-4 is expressed by myeloid progenitor cells.