Targeted disruption of the Runx1/ AML1 gene in mice has demonstrated that it is required for the emergence of definitive hematopoietic cells but that it is not essential for the formation of primitive erythrocytes. These findings led to the conclusion that Runx1 is a stage-specific transcription factor acting only during definitive hematopoiesis. However, the zebrafish and Xenopus homologs of Runx1 have been shown to play roles in primitive hematopoiesis, suggesting that mouse Runx1 might also be involved in the development of primitive lineages. In this study, we show that primitive erythrocytes in Runx1(-/-) mice display abnormal morphology and reduced expression of Ter119, Erythroid Kruppel-like factor (EKLF, KLF1), and GATA-1. These results suggest that mouse Runx1 plays a role in the development of both primitive and definitive hematopoietic cells.