Although there are several markers available for the identification of endoderm derivatives such as the lung, pancreas, liver, and intestine, there are still very few surface markers available for the prospective isolation of the definitive endoderm. Among these, CXCR4 has been used in combination with E-cadherin as a cell surface marker to identify the definitive endoderm. However, CXCR4 expression decreases in late gut epithelium. Here we report a gene, Decay Accelerating Factor (DAF1/CD55), as a candidate surface marker for the identification of the early and late definitive endoderm. Daf1 is expressed in the definitive endoderm and mesoderm in early embryo at E8.5. Flow cytometry analysis of ES cells-derived differentiated cells revealed that DAF1-expressing cells also expressed CXCR4. Moreover, DAF1 expression is maintained until differentiation day 12 in ES cell-derived definitive endoderm cells. Analysis of the Pdx1/GFP-positive cells in E9.5 embryos and ES cell-derived cells with anti-DAF1 revealed that most Pdx1-GFP cells expressed DAF1. These results suggest that DAF1, when used in combination with E-cadherin, is useful for prospective identification of the definitive endoderm cells.