Expression of the αvβ3 integrin is required for normal osteoclast function. We previously showed that an evolutionary conserved NFATc1 binding site is required for RANKL induction and NFATc1 transactivation of the human β3 promoter. The mechanism conferring specificity for RANKL induction and NFATc1 transduction of the β3 gene in osteoclast differentiation is unclear since NFATc1 is expressed and activated in numerous cell types that do not express the β3 gene. PU.1 is an ETS family transcription factor in myeloid cells associated with expression of various osteoclast genes. The present study investigates the role of NFATc1 in concert with PU.1 in osteoclast-specific transcription of the mouse β3 integrin gene. The mouse β3 promoter was transactivated by NFATc1 in RAW264.7 cells and deletion or mutation of either of the conserved NFAT and PU.1 binding sites abrogated transactivation. NFATc1 transactivation of the mouse β3 promoter was specifically dependent on co-transfected PU.1 in HEK293 cells, to the exclusion of other ETS family members. Direct binding of NFATc1 and PU.1 to their cognate sequences was demonstrated by EMSA and NFATc1 and PU.1 occupy their cognate sites in RANKL-treated mouse marrow precursors in chromatin immuno-precipitation (ChIP) assays. TAT-mediated transduction with dominant-negative NFATc1 dose-dependently blocked endogenous expression of the mouse β3 integrin and the formation of TRAP positive multinucleated cells in RANKL-treated mouse macrophages. These data provide evidence that NFATc1, in concert with PU.1, are involved in regulation of β3 integrin expression during osteoclast differentiation and suggest that PU.1 confers specificity to the NFATc1 response to macrophage lineage cells. J. Cell. Physiol. 215: 636–644, 2008. © 2008 Wiley-Liss, Inc.