E14 ES cells were induced to differentiate as EBs and harvested at the days indicated. For sorting, EBs were dispersed to single cells and stained with anti-Flk1 and anti-Podxl antibodies. (A) Flow cytometric analysis showing subdivision of Flk1+ differentiating ES cells based on expression of Podxl. Boxes highlight the clear separation of Flk1-SP and DP populations at d4.75. (B) Representative flow cytometric analysis of sorted populations after re-aggregation and culture. Differentiating EBs were harvested at the indicated times, dissociated and subjected to antibody staining and FACS. Sorted populations (DN, Flk1-SP, Podxl1-SP and DP) were reaggregated and cultured for 20hr and then analyzed using flow cytometry. The new cell populations formed during re-culture are boxed. The d3.5 DP population was too small to analyze and is, therefore, not represented in this figure. This experiment was repeated three times, with comparable results.