AbstractThe transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) regulates the differentiation of a variety of cell types. Here, the role of C/EBPβ expressed by bone marrow mesenchymal stromal cells (BMMSCs) in B-cell lymphopoiesis was examined. The size of the precursor B-cell population in bone marrow was reduced in C/EBPβ-knockout (KO) mice. When bone marrow cells from C/EBPβ-KO mice were transplanted into lethally irradiated wild-type (WT) mice, which provide a normal bone marrow microenvironment, the size of the precursor B-cell population was restored to a level equivalent to that generated by WT bone marrow cells. In coculture experiments, BMMSCs from C/EBPβ-KO mice did not support the differentiation of WT c-Kit+ Sca-1+ Lineage− hematopoietic stem cells (KSL cells) into precursor B cells, whereas BMMSCs from WT mice did. The impaired differentiation of KSL cells correlated with the reduced production of CXCL12/stromal cell-derived factor-1 by the cocultured C/EBPβ-deficient BMMSCs. The ability of C/EBPβ-deficient BMMSCs to undergo osteogenic and adipogenic differentiation was also defective. The survival of leukemic precursor B cells was poorer when they were cocultured with C/EBPβ-deficient BMMSCs than when they were cocultured with WT BMMSCs. These results indicate that C/EBPβ expressed by BMMSCs plays a crucial role in early B-cell lymphopoiesis. Stem Cells 2014;32:730–740