Macrophages differentiate into osteoclasts in response to the critical cytokine RANKL. However, the efficiency of RANKL-mediated osteoclastogenesis can be profoundly influenced by various cytokines. While studies describing the isolated effects of particular cytokines on osteoclastogenesis have been performed, combinatorial effects of cytokines have not been addressed routinely due to the absence of an efficient assay system. To study the effects of cytokine combinations on osteoclast formation, we performed in vitro assays using either the RAW293 cell line or primary murine splenic macrophages as osteoclast precursors. Using a multiparameter cytokine plating method, we analyzed osteoclastogenesis in response to multiple combinations of the following inflammation-related cytokines: RANKL, IFN-gamma, TNF-alpha, IL-1beta, IL-6, IL-10. We further investigated the role of T-cell-related cytokine combinations on osteoclastogenesis by measuring osteoclast area in response to RANKL with IFN-gamma, IL-2, IL-4, IL-6, TGF-ss, and TNF-alpha. Treatments with RANKL, TNF-alpha, and TGF-ss induced maximal osteoclast formation, suggesting a role for these cytokines in the most aggressive forms of inflammatory bone loss. TNF-alpha alone, however, was unable to induce osteoclast formation in the absence of RANKL despite co-administration of other proinflammatory cytokines. IFN-gamma was a potent inhibitor under all conditions, implicating T cells and NK cells in osteoclast inhibition. These studies demonstrate a rapid screening approach for identifying the potential collective effects of multiple factors on osteoclastic bone resorption.