Oct4 is a POU domain homeobox gene, expressed in undifferentiated embryonal carcinoma and embryonic stem cells and is quickly down-regulated upon induction of differentiation. Transcriptional repression of Oct4 is followed by pronounced epigenetic changes on the regulatory region of the gene. Oct4 has a long upstream regulatory region of about 2,600 bp, consisting of proximal enhancer (PE), distal enhancer (DE), and proximal promoter (PP). In this study, we induced differentiation of a human embryonic carcinoma cell line, NT2, under two different adherent and non-adherent culture conditions, and compared histone modifications as the epigenetic marks on the regulatory region of Oct4 gene after 3 days of differentiation. Using chromatin immunoprecipitation coupled with real-time PCR technique, it was shown that the after induction of differentiation the repressive epigenetic marks of hypoacetylation and methylation on lysine-9 of histone H3 occurred very effectively on the upstream of Oct4, especially in PP region. Also, comparing the two culturing systems it was shown that methylation of lysine-9 of H3 histone was more drastic in PE region of adherent cells rather than suspension cells. This epigenetic profile was in agreement with the difference observed in the expression level of Oct4 in these two culturing systems. The current study clearly shows the effective role of cell culture condition on the epigenetic regulation of gene expression.