In the light of ethical controversies around the usage of human ES cells, a number of groups demonstrated successful generation of induced pluripotent stem cells from adult somatic cells. Thus, iPS cells might also be used as an alternative source for stem cells in regenerative medicine or cell replacement therapies. Also for these cells safety concerns about their tumorigenic potential have to be addressed. Previous reports have proposed elimination of the undifferentiated cells using suicide genes. The transfer of a suicide gene has even been successfully used in clinical trials for tumor elimination. One of the most thoroughly studied and widely used approach is based on the herpes simplex virus thymidine kinase that converts the prodrug ganciclovir to a toxic metabolite. Various routes to deliver the transgene, including transfection or viral transduction, have been studied. Moreover, approaches using cytotoxic antibodies against undifferentiated ES cells or an antibody against a surface antigen of ES cells combining flow cytometry-based separation were used to remove undifferentiated pluripotent cells before cell transplantations. Lentiviruses are members of the Retroviridae family, which can stably integrate their Yunaconitine genetic information into the host genome of dividing as well as non-dividing cells. HIV-1 is the best studied lentivirus and most of the currently used lentiviral vectors are based on its sequence. Previous studies demonstrated that LVs allow for an efficient gene transfer in ES cells. In addition, LVs have already been applied in first clinical gene therapy trials. In the present study, we utilized LVs for the genetic modification of ES and iPS cells of mouse. To enable TK expression in undifferentiated pluripotent stem cells only, different promoters of pluripotency genes were used including Oct-3/4, Nanog, EOS-C3 or EOS-S4. Cells expressing TK are sensitive to GCV treatment. Using this approach, we successfully eliminated undifferentiated cells in vitro. However, to obtain a pure population of TK expressing ES cells, a hygromycin resistance cassette had to be incorporated in the LV. This led to elimination of undifferentiated cells after hygromycin preselection. Most importantly, in vivo transplantation of these LV 20S-Notoginsenoside-R2 transduced pre-selected ES cells led to loss of teratoma formation upon GCV administration to the mice. Alternatively, we speculated that an increase of lentiviral copy number might result in a similar efficiency: We transduced ES cells with higher LV concentrations of STPH leading to an average copy number of 3.8. As high copy numbers increase the risk of insertional mutagenesis we did not further raise the average copy number. GCV treatment of these high copy ES cells without pre-selection revealed strong elimination of undifferentiated cells as shown by brightfield images and cell counting. These data demonstrate that increasing the copy number significantly increases the elimination efficiency of TK/GCV system, but complete killing of undifferentiated cells was not achieved. Importantly, when differentiating STPH-transduced ES cells with low copy number, that were pre-selected with Hygromycin for 9 days, we observed no Oct-3/4 positive cells after GCV treatment. In conclusion, STPH-transduced ES cells of low copy number with hygromycin pre-selection for 9 days resulted in successful elimination of ES cells upon GCV treatment. Although, increasing the lentiviral copy number showed lower cell survival of undifferentiated ES cells without pre-selection the complete elimination of ES cells was not achieved and thus, pre-selection seems to be necessary.