• RANKL deficiency in mice increases the frequency of skeletal stem cells (SSCs) but impairs their differentiation along the osteochondrogenic lineage, independent of osteopetrosis.
• Pharmacological blockade of RANKL with Denosumab reduces the osteogenic capacity of human bone marrow-derived stromal cells (hBMSCs) in vitro.
• RANKL is identified as a novel intrinsic regulatory factor of skeletal stem and progenitor cells, with potential translational implications for regenerative medicine.
• The study provides the first characterization of skeletal progenitor cells in severe recessive osteopetrosis mouse models, highlighting RANKL's role beyond osteoclast regulation.