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The 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. S tem  C ells   2014;32:730–740

CCAAT/Enhancer‐Binding Protein β Expressed by Bone Marrow Mesenchymal Stromal Cells Regulates Early B‐Cell Lymphopoiesis