Dental follicle cells (DFCs) can be artificially differentiated into mineralizing cells. With a dexamethasone-based differentiation protocol, transcription factors ZBTB16 and NR4A3 are highly upregulated but Runx2 and other osteogenic marker genes are not. Previous studies have suggested the involvement of a Runx2-independent differentiation pathway. The objective of this study is to further elucidate this mechanism. Differentiation of DFCs was examined by alkaline phosphatase (ALP) staining and ALP activity measurement, by Alizarin Red S staining and by real-time reverse transcription plus the polymerase chain reaction. ZBTB16 was overexpressed by using a transient transfection method. Resulting genome-wide gene expression changes were assessed by microarray. ZBTB16 and Runx2 were inhibited by short interfering RNA transfection. Promoter binding of ZBTB16 was evaluated by chromatin immunoprecipitation. Downregulation of Runx2 had no effect on dexamethasone-induced differentiation but was effective on BMP2-induced differentiation. Downregulation of ZBTB16, however, impaired dexamethasone-induced differentiation. Genes that were upregulated by dexamethasone induction were also upregulated by ZBTB16 overexpression. Genes that were not upregulated during dexamethasone-induced differentiation were also not regulated by ZBTB16 overexpression. ZBTB16 bound directly to the promoter regions of osterix and NR4A3 but not that of Runx2. Overexpression of ZBTB16 led to changes in the gene expression profile, whereby upregulated genes were overrepresented in osteogenesis-associated biological processes. Our findings suggest that, in DFCs, a Runx2-independent differentiation mechanism exists that is regulated by ZBTB16.