Culturing human Mesenchymal stromal cells (hMSCs) in vitro in hypoxic conditions resulted in reduced senescence, enhanced pluripotency and altered proliferation rate. It has been known that in vitro hypoxia affects expression of cell surface proteins. However, the impact of culture surfaces on the hypoxia-regulated genes (HRG) have not yet been reported. This study utilized Next-Generation sequencing to analyse the changes in the gene expression levels of HRG for hMSCs cultured on different culture surfaces. The samples, which were cultured on four different synthesized surfaces (treatments) and tissue culture plate (control), resulted in a difference in growth rate. The sequencing results revealed that the transcription of a number of key genes involved in regulating hypoxic functions were significantly altered, including HIF2A, a marker for potency, differentiation, and various cellular functions. Significant alternations in the expression levels of previously reported oxygen-sensitive surface proteins were detected in this study, some of which closely correlate with the expression levels of HIF2A. Our analysis of the hMSCs transcriptome and HRG mapped out a list of genes encoding surface proteins which may directly regulate or be regulated by HIF2A. The findings from this study showed that culture surfaces have an impact on regulating the expression profile of HRG. Therefore, novel culture surfaces may be designed to selectively activate HIF2A and other HRG and pathways under in vitro normoxia. The understanding of the crosstalk between the regulating genes of hypoxia and culture surfaces may be utilized to strengthen desired hypoxic functions.