The precise mechanism of osteolysis induced by tumors infiltrating into the bone remains unclear. The main hypothesis is that tumor cells generate receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor-alpha (TNF-alpha), or other molecules that activate the expression of RANKL in osteoblasts, osteocytes, or bone marrow stromal cells. Administration of bisphosphonates or anti-RANKL antibody drugs, which suppress systemic bone resorption, prevents osteolysis induced by tumors infiltrating into the bone. However, these therapeutic agents may cause medication-related osteonecrosis of the jaw. In this study, we found a novel tumor-associated osteoclastogenesis pathway in osteoclast precursor cells. Co-culture with human oral squamous cell carcinoma cells, 3A or NEM, or culture with each of their conditioned medium induced many osteoclasts from osteoclast precursor cells, which were generated by a 24-h pretreatment of RANKL or TNF-alpha. Osteoprotegerin, a decoy RANKL receptor, denosumab, an anti-RANKL antibody drug, and infliximab, an anti-TNF-alpha antibody drug, did not prevent this tumor-associated osteoclastogenesis. Quantitative RT-PCR analysis showed that the expression of NFATc1 was decreased in this tumor-associated osteoclastogenesis, which was suggested to be independent of NFATc1. These results revealed a novel pathway for tumor-associated osteoclastogenesis, which may be a new therapeutic target for osteolysis induced by tumors infiltrating into the bone without affecting systemic bone metabolism. (C) 2018 Elsevier Inc. All rights reserved.