In this research, we use mouse embryonic fibroblasts as feeder layers. To eliminate the influence of serum and mouse embryonic stem cells(ESCs) conditioned medium(ESCCM) on self-renewal of sheep embryonic stem-like cells, knockout serum replacement(KSR) was used to replace serum, then supplanted with ESCCM for the isolation and cloning of sheep embryonic stem-like cells. We found when inner cell masses(ICMs) cultured in the control group with medium supplanted with fetal bovine serum(FBS), sheep ES-like cells could not survive for more than 3 passages. However, sheep embryonic stem-like cells could remain undifferentiated for 5 passages when cultured in the medium that FBS was substituted by KSR. The result indicates that KSR culture system was more suitable for the isolation and cloning of sheep embryonic stem-like cells compared to FBS culture system. Finally we applied medium with 15% KSR as basic medium supplanted with 40% ESCCM as a new culture system to isolate sheep embryonic stem-like cells, we found one embryonic stem-like cell line still maintained undifferentiating for 8 passages, which characterized with a normal and stable karyotype and high expression of alkaline phosphatase. These results suggest that it is suitable to culture sheep ICM in the new culture system with 15% KSR as basic medium and supplanted with 40% ESCCM, which indicated that mouse ES cells might secrete factors playing important roles in promoting sheep ES-like cells’ self-renewal.