The development of blood cells from hematopoietic stem cells in the bone marrow is dependent on communication with bone marrow stroma cells, making these cells central for the appropriate regulation of hematopoiesis. To identify transcription factors that may play a role in gene regulation in stroma cells, we performed comparative gene expression analysis of fibroblastic NIH3T3 cells, unable to support hematopoiesis in vitro, and OP-9 stroma cells, highly efficient in this regard. These experiments revealed that transcription factors of the early B cell factor (EBF) family were highly expressed in OP-9 cells as compared with the NIH3T3 cells. To identify potential targets genes for EBF proteins in stroma cells, we overexpressed EBF in fibroblasts and analyzed the pattern of induced genes by microarray analysis. This revealed that EBF was able to up-regulate expression of among others the Cxcl12, Ccl9, and Periostin genes. The identification of relevant promoters revealed that they all contained functional EBF binding sites able to interact with EBF in OP-9 cells. Furthermore, ectopic expression of a dominant negative EBF protein or antisense EBF-1 RNA in OP-9 stroma cells resulted in reduced expression of these target genes. These data suggest that EBF proteins might have dual roles in hematopoiesis acting both as intrinsic regulators of B-lymphopoiesis and as regulators of genes in bone marrow stroma cells.