Oct4 is a key transcription factor to maintain self-renewal and undifferentiated state of embryonic stem cells. Site 2A located between -2,546 and -2,530 bp and site 2B between -2,500 and -2,486 bp of human Oct4 gene were shown to be sufficient for inducing Oct4 gene expression in embryonic stem cells. Site 2B contains octamer element capable of binding to factor Oct4 and sox element capable of binding to factor Sox2. So far, little is known about the molecular mechanisms for the control of growth and differentiation of adult stem cells including bone marrow-derived mesenchymal stem cells (BM-MSCs), and it is important to understand how Oct4 expression is regulated in BM-MSCs. This study showed that Oct4 and Sox2 genes were expressed in undifferentiated BM-MSCs and BM-MSCs on day 7 but not on days 14 and 21 following osteogenic induction. Site 2A of Oct4 gene, not site 2B, activated the expression of reporter genes luciferase and enhanced green fluorescent protein in undifferentiated BM-MSCs but not in BM-MSCs following osteogenic differentiation. These data demonstrate that site 2A is sufficient for inducing the expression of Oct4 gene in BM-MSCs, and site 2B is not required. Electrophoretic mobility shift assay showed the 2 shifted bands with site 2B probe and the addition of Oct4 and Sox2 antibodies did not supershift these 2 bands. As probes containing mutated octamer and sox elements of site 2B still gave the same 2 shifted bands, it was concluded that they did not result from the binding to site 2B probe by factors Oct4 and Sox2. These bands may be due to the binding of 2 unknown transcription factors.