Interleukin‐34 (IL‐34) is a newly discovered regulator of myeloid lineage differentiation, proliferation, and survival, acting via the macrophage‐colony stimulating factor receptor (M‐CSF receptor, c‐fms). M‐CSF, the main ligand for c‐fms, is required for osteoclastogenesis and has been already identified as a critical contributor of the pathogenesis of giant cell tumours of bone (GCTs), tumours rich in osteoclasts. According to the key role of M‐CSF in osteoclastogenesis and GCTs, the expression of IL‐34 in human GCTs was first assessed. Quantitative analysis of IL‐34 mRNA expression in 14 human GCTs revealed expression of this cytokine in GCTs as well as M‐CSF and c‐fms. Immunohistochemistry demonstrated that osteoclast‐like cells exhibited a huge immunostaining for IL‐34 and that mononuclear stromal cells were slightly positive for this protein. In contrast to osteoblasts, bone‐resorbing osteoclasts showed very strong staining for IL‐34, suggesting its potential role in the pathogenesis of GCTs by facilitating osteoclast formation. The role of IL‐34 in osteoclastogenesis was then studied in murine and human models. IL‐34 was able to support RANKL‐induced osteoclastogenesis in the absence of M‐CSF in all models. Multinucleated cells generated in the presence of IL‐34 and RANKL expressed specific osteoclastic markers and resorbed dentine. IL‐34 induced phosphorylation of ERK 1/2 and Akt through the activation of c‐fms, as revealed by the inhibition of signalling by a specific c‐fms tyrosine kinase inhibitor. Furthermore, IL‐34 stimulated RANKL‐induced osteoclastogenesis by promoting the adhesion and proliferation of osteoclast progenitors, and had no effect on osteoclast survival. Overall, these data reveal that IL‐34 can be entirely substituted for M‐CSF in RANKL‐induced osteoclastogenesis, thus identifying a new biological activity for this cytokine and a contribution to the pathogenesis of GCTs. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.