The inconsistency of efficiency in murine embryonic stem cell (ESC) production might be associated with the differences in preparation and cryopreservation of the feeder cell layer. As the cryopreservation of mouse embryonic fibroblast (MEF) declined the quality of MEF as feeder cell layer, an effective protocol should be determined to produce murine ESC on frozen-thawed feeder cell layer as efficient on fresh feeder cell layer. Under appropriate culture conditions, isolated inner cell mass (ICM) of murine blastocyst will form ESC and be maintained in undifferentiated state. Therefore, the aims of this study were to determine the most optimum freezing density and equilibration duration for cryopreserving MEF feeder cell layer and to determine the effect of fresh and frozen-thawed feeder cell layer on murine ESC production. Freezing density of 5×106 cells÷mL gave a significantly higher viability rate than 0.5×106 cells÷mL (68.08% vs. 59.78%, p<0.05) and comparable with 2×106 cells÷mL. The viability rates of frozen-thawed MEF derived from 15 minutes equilibration was significantly higher than 20 hours equilibration (79.4% vs. 68.08, p<0.05). There were no significant differences between fresh and frozen-thawed MEF feeder cell layer for percent successful attachment of blastocysts, consecutive passages of murine ESC up to passage 3. In conclusion, freezing density of 5×106 cells÷mL and 15 minutes equilibration duration are optimizing the cryopreservation of MEF feeder cell layer to subsequently improve the production of murine ESC.