CCAAT/enhancer binding proteins (C/EBP) have been demonstrated to impact directly the development of multiple hematopoietic lineages. However, the role of C/EBPβ in the differentiation of various hematopoietic lineages has not been thoroughly examined. We used primary bone marrow cultures to assess directly the ability of C/EBPβ to influence myelopoiesis. Retroviral expression vectors were used to express C/EBPβ ectopically in murine primary long-term bone marrow cultures. The differentiation potential of these cells was determined using hematopoietic colony assays and differential staining of cells within the cultures. Bone marrow cultures that overexpressed C/EBPβ had fewer myeloid progenitors and a significant increase in the number of granulocytes. The ability of C/EBPβ to alter hematopoiesis in vitro was dependent on the presence of the transcriptional activation domain because LIP, which lacks this functional domain, did not decrease the ability of bone marrow cultures to support myeloid progenitors. These data also show that C/EBPβ influences hematopoiesis by altering stromal cell function rather than the intrinsic developmental potential of myeloid progenitor cells.