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The runt family transcription factor (AML‐3/PEBP2αA1/Cbfa1/RUNX2) plays a crucial role in formation of the mineralized skeleton during embryogenesis and regulates maturation of the osteoblast phenotype. Because steroid hormones and growth factors significantly influence growth and differentiation properties of osteoblasts, we addressed Cbfa1 as a target gene for regulation by dexamethasone (Dex), 1,25(OH)D₃ (vitamin D₃), 17β‐estradiol, and transforming growth factor‐β1 (TGF‐β1). The representation of functional protein levels by Western blot analyses and gel mobility shift assays was examined during the growth and mineralization of several conditionally immortalized human osteoblast cell lines HOB 04‐T8, 03‐CE6, and 03‐CE10, each representing different stages of maturation. In situ immunofluorescence demonstrates Cbfa1 is associated with nuclear matrix in punctate domains, some of which are …