Embryonic stem (ES) cells constitute a very important tool for regenerative medicine today. These ES cells, and human ES cells in particular, are almost all derived from embryos obtained by in vitro fertilization (IVF) and from in vitro culture (IVC); however, such in vitro manipulated embryos often show abnormal genomic imprinting, which can lead to the development of various diseases. Nevertheless, several reports have evaluated ES cells derived from in vitro manipulated embryos. In this study, we established ES cells derived from both in vivo and in vitro developed blastocysts (Vivo ES cells and Vitro ES cells, respectively) to compare the methylation status of imprinted genes and gene expression patterns. At very early passages, Vitro ES cells showed an increase in abnormal genomic imprinting compared to Vivo ES cells. In addition, we found that the gene expression patterns of several methylation related-genes frequently shifted to promote demethylation and to inhibit methylation in early-passage Vitro ES cells. In contrast, at later passages, we found no significant differences between Vivo and Vitro ES cells. In conclusion, it is advisable to use early passage Vivo ES cells whenever feasible, or to select ES cell lines with a normal epigenotype.