The formation of bone resorbing osteoclasts in vivo is orchestrated by cells of the osteoblast lineage such as periodontal ligament fibroblasts that provide the proper signals to osteoclast precursors. Although the requirement of cell-cell interactions is widely acknowledged, it is unknown whether these interactions influence the expression of genes required for osteoclastogenesis and the ultimate formation of osteoclasts. In the present study we investigated the effect of cell-cell interaction on the mRNA expression of adhesion molecules and molecules involved in osteoclast formation in cultures of peripheral blood mononuclear cells (PBMCs) and human primary periodontal ligament fibroblasts, both as solitary cultures and in co-culture. We further analyzed the formation of multinucleated, tartrate resistant acid phosphatase (TRACP) positive cells and assessed their bone resorbing abilities. Interestingly, gene expression of intercellular adhesion molecule-1 (ICAM-1) and of osteoclastogenesis-related genes (RANKL, RANK, TNF-α, and IL-1β) was highly up-regulated in the co-cultures compared to mono-cultures and the 5-10-fold up-regulation reflected a synergistic increase due to direct cell-cell interaction. This induction strongly overpowered the effects of known osteoclastogenesis inducers 1,25(OH)2VitD3 and dexamethasone. In case of indirect cell-cell contact mRNA expression was not altered, indicating that heterotypic adhesion is required for the increase in gene expression. In addition, the number of osteoclast-like cells that were formed in co-culture with periodontal ligament fibroblasts was significantly augmented compared to mono-cultures. Our data indicate that cell-cell adhesion between osteoclast precursors and periodontal ligament fibroblasts significantly modulates the cellular response which favors the expression of osteoclast differentiation genes and the ultimate formation of osteoclasts.