Ash2l promotes H3K4 trimethylation levels through methyltransferase MLL/SET1 complex, which is essential for mouse embryonic development. Ash2l have two isoforms (Ash2l-1 and Ash2l-2) through alternative promoter usage in mouse. However, the mechanism of this gene in mouse embryonic development and the function of different isoforms remain unknown. In this study, we used CRISPR/Cas9 technology to specifically disrupt Ash2l-1 in mice and investigated the role of Ash2l-1 in mouse early embryonic development. Disruption of Ash2l-1 resulted in embryonic lethality at E9.5-10.5. Particularly, E9.5 Ash2l-1-deficient embryos exhibited severe growth defects, including developmental defects of yolk sac. Gene expression profiling showed that Ash2l-1 deficiency affected the expression of specific genes involved in erythropoiesis and vascular formation. CUT&RUN analysis showed that H3K4me3 levels of the promoter of some specific genes involved in erythropoiesis and vascular formation was down-regulated. Taken together, these results indicate that Ash2l-1 regulates gene expression through H3K4me3 and plays an essential role in the early hematopoiesis of mouse yolk sacs.