Post-translational modifications (PTMs) are dynamic processes that regulate cell states by enhancing proteome diversity. However, the overall impact of PTMs on pluripotency exit in porcine embryonic stem cells (pESCs) remains largely unknown. Here, we present a systematic assay to identify E3 ubiquitin ligases for pluripotency exit by using CRISPR/Cas9 pooled screening and identified PIAS4 as a major regulator of pluripotency exit, as the cell differentiation was significantly impaired upon PIAS4 depletion in pESCs. PIAS4 shows a high degree of genomic occupation in promoter regions, particularly in key pluripotency maintenance genes. Moreover, we found that PIAS4 was recruited to the gene promoter marked by H3K4me3 and interacted with lysine demethylase KDM5B via SUMOylation, thereby affecting the stability of KDM5B and further facilitating the regulation of H3K4me3-mediated lineage-specific genes. Together, our findings reveal a regulatory mechanism by which PIAS4 modulates H3K4me3 modification on development-related genes, subsequently influencing pluripotency exit and cell fate commitment by interacting with KDM5B in pESCs.