Embryonic stem cell (ESC) research gave rise to the possibility that stem cell therapy could be used in the treatment of incurable diseases such as neurodegenerative disorders. However, problems related to the tumorigenicity of undifferentiated ESCs must be resolved before such cells can be used in the application of cell replacement therapies. In the present study, we attempted to determine biomarkers that predicted tumor formation of undifferentiated ESCs in vivo. We differentiated mouse ESCs (R1 cell line) into neural lineage using a 5-step method, and evaluated the expression of oncogenes (p53, Bax, c-myc, Bcl2, K-ras), telomerase-related genes (TERT, TRF), and telomerase activity and telomere length during differentiation of ESCs. The expression of oncogenes did not show a significant change during differentiation steps, but the expression of telomerase reverse transcriptase (TERT) and telomerase activity correlated with mouse ESCs differentiation. To investigate the possibility of mouse TERT (mTERT) as a biomarker of tumorigenicity of undifferentiated ESCs, we established mTERT knockdown ESCs using the shRNA lentivirus vector and evaluated its tumorigenicity in vivo using nude mice. Tumor volumes significantly decreased, and appearances of tumor formation in mice were delayed in the TERT-knockdown ESC treated group compared with the undifferentiated ESC treated group. Altogether, these results suggested that mTERT might be potentially beneficial as a biomarker, rather than oncogenes of somatic cells, for the assessment of ESCs tumorigenicity.