As the zinc-finger transcription factor specificity protein 3 (Sp3) has been implicated in the regulation of many hematopoietic-specific genes, we analyzed the role of Sp3 in hematopoiesis. At embryonic day 18.5 (E18.5), Sp3-/- mice exhibit a partial arrest of T-cell development in the thymus and B-cell numbers are reduced in liver and spleen. However, pre-B-cell proliferation and differentiation into immunoglobulin M-positive (IgM+) B cells in vitro are not affected. At E14.5 and E16.5, Sp3-/- mice exhibit a significant delay in the appearance of definitive erythrocytes in the blood, paralleled by a defect in the progression of differentiation of definitive erythroid cells in vitro. Perinatal death of the null mutants precludes the analysis of adult hematopoiesis in Sp3-/- mice. We therefore investigated the ability of E12.5 Sp3-/- liver cells to contribute to the hematopoietic compartment in an in vivo transplantation assay. Sp3-/- cells were able to repopulate the B- and T-lymphoid compartment, albeit with reduced efficiency. In contrast, Sp3-/- cells showed no significant engraftment in the erythroid and myeloid lineages. Thus, the absence of Sp3 results in cell-autonomous hematopoietic defects, affecting in particular the erythroid and myeloid cell lineages.