H3K4me3 is an important epigenetic modification that plays a critical role in maintaining self-renewal of mouse embryonic stem cells (mESCs). H3K4me3 is catalyzed mainly by the mixed lineage leukemia (MLL) methyl-transferase complex. ASH2L, a core subunit of the MLL complex, participates in regulating the open state of chromatin in mESCs. There are two isoforms of the ASH2L protein: ASH2L-1 (80 kDa), which only exists in mouse embryonic fibroblasts and ASH2L-2 (65 kDa), which is the predominant isoform in mESCs. The roles of Ash2l-1 and Ash2l-2 in mESCs have not yet been elucidated. In this study, we established Ash2l-1 ^-/- and Ash2l-2 ^-/- knockout mESCs using CRISPR/Cas9. Alkaline phosphatase (AP) staining, immunofluorescence staining, and qRT-PCR showed that there were no obvious differences on the expression level of AP and pluripotent transcription factors (Nanog, Oct4, sox2 and Klf4) among Ash2l-1 ^-/- mESCs, Ash2l-2 ^-/- mESCs and wild type (WT) mESCs. However, analysis of embryoid body (EB) differentiation showed that the expression level of Snai2 (ectoderm gene) and Gata4 (endoderm gene) in Ash2l-1 ^-/- EBs was significantly lower than that in WT EBs (P<0.01). Western blotting assay revealed that the expression of ASH2L-2 was significantly increased (P<0.01) in Ash2l-1 ^-/- mESCs and vice versa. However, there were no obvious differences on the genomic H3K4me3 level among Ash2l-1 ^-/- mESCs, Ash2l-2 ^-/- mESCs and WT mESCs. These results indicate that there exist compensation effects between Ash2l-1 and Ash2l-2. Bioinformatic analysis predicted that there were three and 16 potential binding sites for pluripotency transcription factors located in the promoter of Ash2l-1 and Ash2l-2, respectively. Theses transcription factors may mediate the compensation effect between Ash2l-1 and Ash2l-2. Collectively, these results indicate that the compensation effects between Ash2l-1 and Ash2l-2 may be involved in the maintenance of mESCs pluripotency and the regulation of genomic H3K4me3.