In this study, we identify and characterize the promoter of the mouse Gas6 gene, a negative regulator of chondrogenic differentiation. We identified two highly conserved regions within the core Gas6 promoter, which are conserved among mouse, human, and rat Gas6 genes, named A-BOX and B-BOX. Basal transcriptional activity was significantly reduced after deletion of either the A-BOX OF the latter half of the B-BOX. After treatment with BMP-2 for 3 days, putative A-BOX Binding Factor(s) B (ABFB) and B-BOX Binding Factor(s) (BBF) were unable to bind to their specific motifs in C3H10T1/2 cells, compared with untreated control cells. In addition, we confirmed binding of an NF-Y site within the core promoter region of mouse Gas6 gene. Taken together, these observations provide insight into the mechanism by which Gas6 is downregulated during mesenchymal stem cells differentiation into chondrocytes. (C) 2008 Elsevier Inc. All rights reserved.