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Introduction: Nowadays, mesenchymal stem cells are touted as suitable cell supply for the restoration of injured bone tissue. The existence of osteogenic differentiation makes these cells capable of replenishing damaged cells in the least possible time. It has been shown that epigenetic modifications, especially DNA methylation, contribute to the regulation of various transcription factors during phenotype acquisition. Hence, we concentrated on the correlation between the promoter methylation and the expression of genes DLX3, ATF4, and FRA1 during osteoblastic differentiation of adipose-derived mesenchymal stem cells in vitro after 21 days. Methods: Adipose-derived mesenchymal stem cells were cultured in osteogenesis differentiation medium supplemented with 0.1 µM dexamethasone, 10 mM β-glycerol phosphate, and 50 µM ascorbate-2-phosphate for 21 days. RNA and DNA extraction was done on days 0, 7, 14, and 21. Promoter methylation and expression levels of genes DLX3, ATF4, and FRA1 were analyzed by methylation-specific quantitative PCR and real-time PCR assays, respectively. Results: We found an upward expression trend with the increasing time for genes DLX3, ATF4, and FRA1 in stem cells committed to osteoblast-like lineage compared to the control group (PDLX3 and ATF4 genes, but not FRA1, over time compared to the non-treated control cells (PDLX3 and ATF4 and methylation rate (PConclusion: In particular, osteoblastic differentiation of adipose-derived mesenchymal stem cells enhances DLX3 and ATF4 transcriptions by reducing methylation rate for 21 days.