There is evidence demonstrating the regulation of microRNAs in a myriad of vascular biology events such as cardiogenesis, but the role of miRNA in controlling human embryonic stem cell fate in differentiation towards the endothelial lineage remains to be studied. To study this issue, we designed and developed a differentiation culture model to generate endothelial cells from undifferentiated human embryonic stem cells. We aim to investigate the in vivo therapeutic effects of differentiated endothelial cells derived from human stem cells as well as determining the role of miRNA/s involved in vascular lineage differentiation. Undifferentiated human embryonic stem cells were firstly cultured in differentiation conditions to derive endothelial cells. Differentiated day 9 stem cell-derived endothelial cells expressed specific endothelial markers such as CD31 and CD144. At day 17 of differentiation, flow cytometry analysis showed that 34.1% of the heterogeneous differentiated human stem cells were CD146 positive. This population of cells was sorted for CD146 and expanded in vitro. Expanded CD146 positive cells were capable of ac-LDL uptake, binding to Lectin and formation of vascular structures on Matrigel, suggesting that expanded CD146 positive cell are functional endothelial cells. Determination of potential role of miRNA involved in early endothelial development was next carried out using miRNA array expression profiling in the differentiating human embryonic stem cells. Five potential upregulated miRNA, were selected from the miRNA array analysis and further analysis found that miR-150* and miR-200c were crucial in vascular endothelial lineage differentiation. It was found that the targets of these miRNAs in stem cell differentiation involved several proteins/transcription factors. Thus, we established an effective model to derive endothelial cells in vitro and demonstrated the involvement of miR-150* and miR-200c in human stem cell differentiation, implicating a potential usefulness for stem cell therapy in the future.