We have previously shown that mice lacking the TSH receptor (TSHR) exhibit osteoporosis due to enhanced osteoclast formation. The fact that this enhancement is not observed in double-null mice of TSHR and TNFα suggests that TNFα overexpression in osteoclast progenitors (macrophages) may be involved. It is unknown how TNFα expression is regulated in osteoclastogenesis. Here, we describe a receptor activator for nuclear factor-κB ligand (RANKL)-responsive sequence (CCG AGA CAG AGG TGT AGG GCC), spanning from −157 to −137 bp of the 5′-flanking region of the TNFα gene, which functions as a cis-acting regulatory element. We further show how RANKL treatment stimulates the high-mobility group box proteins (HMGB) HMGB1 and HMGB2 to bind the RANKL-responsive sequence and up-regulates TNFα transcription. Exogenous HMGB elicits the expression of cytokines, including TNFα, as well as osteoclast formation. Conversely, TSH inhibits the expression of HMGB and TNFα and the formation of osteoclasts. These results suggest that HMGB play a pivotal role in osteoclastogenesis. We also show a direct correlation between the expression of HMGB and TNFα and osteoclast formation in TSHR-null mice and TNFα-null mice. Taken together, we conclude that HMGB and TNFα play critical roles in the regulation of osteoclastogenesis and the remodeling of bone.