For pluripotent stem cell (PSC)-based regenerative therapy against diabetes, the differentiation efficiency to pancreatic lineage cells needs to be improved based on the mechanistic understanding of pancreatic differentiation. Here, we aimed to elucidate the molecular mechanisms underlying pancreatic endoderm differentiation by searching for factors that regulate a crucial pancreatic endoderm marker gene, NKX6.1. Unbiasedly screening an siRNA knockdown library, we identified a candidate transcription factor, HHEX.HHEXknockdown suppressed the expression of another pancreatic endoderm marker gene,PTF1A, as well asNKX6.1, independently ofPDX1, a known regulator ofNKX6.1expression. In contrast, the overexpression ofHHEXupregulated the expressions ofNKX6.1andPTF1A. RNA-seq analysis showed decreased expressions of several genes related to pancreatic development, such asNKX6.1,PTF1A,ONECUT1andONECUT3, inHHEXknockdown pancreatic endoderm. These results suggest that HHEX plays a key role in pancreatic endoderm differentiation.