Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocyst and differentiate into all three embryonic germ layers: ectoderm, endoderm, and mesoderm. In this study, ESCs are derived from Hand Made Cloning (HMG) blastocysts and their efficiencies compared to ESCs derived from In Vitro Fertilization (IVF) embryos. Feeder layer was used for ESCs culture, and culture medium consisting of Knockout- Dulbecco’s Modified Eagle’s Medium (Ko-DMEM) supplemented with Knockout Serum Replacement (KSR), Leukemia Inhibitory Factor (LIF), Basic Fibroblast Growth Factor-2 (FGF-2), L-glutamine, nonessential amino acids and gentamicin. The cell surface antigens used for characterization were the SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81 and the pluripotency markers were NANOG, OCT3/4 and SOX2. Results showed that, the growth rate of ESCs colonies in ESCs from IVF embryos was significantly higher than ESCs from HMG embryos (120% compared with 65%, respectively). Not only real-time PCR results revealed the same expression level of SOX2, OCT3/4 and cMYC between them, but also ESCs from HMG embryos resulted to higher expression of NANOG. Both of ESCs groups maintain in pluripotency state for more than two years and differentiated to the different types of cells like neuron, epithelial, lipid and muscle cells.