Differentiation of bone-resorbing osteoclasts from hematopoietic precursors depends upon expression of the cytokine receptor activator of NFkappaB ligand (RANKL) by fibroblastic stromal cells, which some evidence suggests are of the osteoblast lineage. We have shown previously that hormonal-responsiveness of the murine RANKL gene is mediated in part by a distal enhancer that binds Runx2, a transcription factor required for commitment to the osteoblast lineage, supporting the idea that osteoclast-supporting stromal cells may be osteoblasts or their progenitors. However, in this study we demonstrate that parathyroid hormone (PTH) stimulation of RANKL in mice is not affected by a significant reduction in the number of osteoblasts. Consistent with this, neither Runx2, nor Cbfb, a binding partner essential for Runx activity, are required for basal or PTH-stimulated RANKL expression in fibroblastic stromal cell models. Nonetheless, RANKL responsiveness to PTH was elevated in cultured calvaria cells expressing high levels of osterix, another transcription factor required for osteoblast differentiation, and this was associated with elevated PTH receptor expression. The responsiveness of RANKL to 1,25-dihydroxyvitamin D(3) was not elevated in the osterix-expressing cells. Together, these results suggest that commitment to the osteoblast lineage is not a requirement for RANKL gene transcription in fibroblastic stromal cells but may enhance responsiveness of this gene to specific hormones via control of their receptors.