The optimization of in vitro culture conditions for embryonic stem cells (ESCs) is a matter of critical importance; a prompt supply of a sufficient population of cells that retain their pluripotency capabilities must be secured in order to make possible future cell therapies. Despite a number of reports asserting that a variety of cytokines, signaling ligands, and small molecules can help in maintaining the pluripotency of ESCs, mammalian feeder cells continue to be broadly accepted as the method of choice for ESC cultures. This appears to be because mammalian feeder cells seem to produce some as-yet-unidentified factor that makes them very effective as feeder cells. In this study, we investigated wild-type p53 inducible phosphatase (Wip1), the knockdown of which increases Wnt inhibitory factor-1 expression, in its feeder functions toward mouse embryonic stem cells, lowering the effect of Wnt, one of key signaling in maintaining stemness of ESCs. For this purpose, Wip1 was stably expressed in mouse embryonic fibroblast cell line (STO) using retro-viral gene delivery system and then the function as a feeder cell was monitored either with or without leukemia inhibitory factor (LIF) in culture medium. We demonstrated that mouse embryonic stem cells grown with Wip1 expressing STO showed higher alkaline phosphatase activity and sustained Oct-4 expression level even under LIF deprivation condition compared to both control and Wip1 phosphatase activity dead mutant expressing STO. These results imply that Wip1 phosphatase activity in feeder cells is important to retain pluripotency of mouse embryonic stem cells under LIF deprivation conditions. These results indicate that genetically engineered feeder cells such as Wip1 expressing cell lines, are alternative strategy for the optimization of maintenance and expansion of mouse embryonic stem cells.