Differentiation of embryonic stem cells (ESCs) into insulin-producing cells for cell replacement therapy of diabetes mellitus comprises the stepwise recapitulation of in vivo developmental stages of pancreatic organogenesis in an in vitro differentiation protocol. The chemical compounds IDE-1 and (-)-indolactam-V can be used to direct mouse and human ESCs through these stages to form definitive endoderm via an intermediate mesendodermal stage and finally into pancreatic endoderm. Cells of the pancreatic endoderm express the PDX1 transcription factor and contribute to all pancreatic cell types upon further in vitro or in vivo differentiation. Even though this differentiation approach is highly effective and reproducible, it generates heterogeneous populations containing PDX1-expressing pancreatic progenitors amongst other cell types. Thus, a technique to separate PDX1-expressing cells from this mixture is very desirable. Recently it has been reported that PDX1-positive pancreatic progenitors, derived from human embryonic stem cells, express the surface marker CD24. Therefore were subjected mouse and human ESCs to a small molecule differentiation approach and the expression of the surface marker CD24 was monitored in undifferentiated cells, cells committed to the definitive endoderm and cells reminiscent of the pancreatic endoderm. We observed that both mouse and human ESCs expressed CD24 in the pluripotent state, during the whole process of endoderm formation and upon further differentiation towards pancreatic endoderm. Thus CD24 is not a suitable cell surface marker for identification of PDX1-positive progenitor cells.