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Embryonic stem cells (ESCs) derived from outbred mice which share several genetic characteristics similar to humans have been requested for developing stem cell-based bioengineering techniques directly applicable to humans. Here, we report the generation of ESCs derived from the inner cell mass of blastocysts retrieved from 9-week-old female outbred ICR mice mated with 9-week-old male outbred ICR mice ( ICR ESCs). Similar to those from 129/Ola mouse blastocysts ( E14 ESCs), the established ICR ESCs showed inherent characteristics of ESCs except for partial and weak protein expression and activity of alkaline phosphatase. Moreover, ICR ESCs were not originated from embryonic germ cells or pluripotent cells that may co-exist in outbred ICR strain-derived mouse embryonic fibroblasts ( ICR MEFs) used for deriving colonies from inner cell mass of outbred ICR mouse blastocysts. Furthermore, instead of outbred ICR MEFs, hybrid B6CBAF1 MEFs as feeder cells could sufficiently support in vitro maintenance of ICR ESC self-renewal. Additionally, ICR ESC-specific characteristics (self-renewal, pluripotency, and chromosomal normality) were observed in ICR ESCs cultured for 40th subpassages (164 days) on B6CBAF1 MEFs without any alterations. These results confirmed the successful establishment of ESCs derived from outbred ICR mice, and indicated that self-renewal and pluripotency of the established ICR ESCs could be maintained on B6CBAF1 MEFs in culture.