- Osteoclasts are multinucleated, bone-resorbing cells formed via fusion of monocyte progenitors, a process triggered by prolonged stimulation with RANKL, the osteoclast master regulator cytokine. Monocyte fusion into osteoclasts has been shown to play a key role in bone remodeling and homeostasis; therefore, aberrant fusion may be involved in a variety of bone diseases. Indeed, research in the last decade has led to the discovery of genes regulating osteoclast fusion; yet the basic cellular regulatory mechanism underlying the fusion process is poorly understood. Here, we applied a novel approach for tracking the fusion processes, using live-cell imaging of RANKL-stimulated and non-stimulated progenitor monocytes differentially expressing dsRED or GFP, respectively. We show that osteoclast fusion is initiated by a small (~ 2.4%) subset of precursors, termed “fusion founders”, capable of fusing either with other founders or with non-stimulated progenitors (fusion followers), which alone, are unable to initiate fusion. Careful examination indicates that the fusion between a founder and a follower cell consists of two distinct phases: an initial pairing of the two cells, typically lasting 5–35 min, during which the cells nevertheless maintain their initial morphology; and the fusion event itself. Interestingly, during the initial pre-fusion phase, a transfer of the fluorescent reporter proteins from nucleus to nucleus was noticed, suggesting crosstalk between the founder and follower progenitors via the cytoplasm that might directly affect the fusion process, as well as overall transcriptional regulation in the developing heterokaryon.