CD34 is a cell-surface glycoprotein expressed in a developmental, stage-specific manner by bone marrow stem and progenitor cells. In this study we explored a possible role for c-Myb in CD34 regulation during developmental hematopoiesis. The results indicate that c-Myb can induce CD34 expression in hematopoietic and nonhematopoietic cells, and that murine CD34 promoter activity is enhanced in myeloid cells transgenic for c-Myb. To test whether c-Myb is necessary for CD34 expression during developmental hematopoiesis in vitro, c-Myb-null D3 embryonic stem (ES) cells were analyzed for their ability to develop CD34+ hematopoietic cells in vitro. CD34 promoter activity in transient transfections and CD34 upregulation during ES cell differentiation into embryoid bodies was identical in wild-type and c-Myb-null ES cells, indicating that c-Myb is not required for CD34 expression. CD34 protein is expressed on both hematopoietic and endothelial cells of the E8.5 blood islands during the development of c-Myb-null embryos, and expression is nearly identical in wild-type and c-Myb-null embryos. However, in E12.5 c-Myb-null embryos, the majority of identifiable CD34+ cells in the developing liver are endothelial rather than hematopoietic, which is consistent with the absence of colony-forming units in c-Myb-null embryos and developing ES cells. These data indicate that c-Myb is not required for CD34 expression in endothelial or primitive hematopoietic cells in the yolk sac, but is necessary for definitive hematopoiesis.