Quantitative real-time RT-PCR and FACS expression analysis of key hematoendothelial genes in differentiating hEBs. Human-specific PCR primers (Table 1) were used to amplify indicated target genes. Levels of gene expression differences for (A) hematoendothelial surface markers or (B) hematopoietic regulatory transcription factors were calculated using the 2^–ΔΔCT method as described in “Materials and methods,” based on the C_T (threshold curve) for each target gene and internal normalizations with actin. Values of fold change in expression are relative to baseline expression levels in FACS-sorted populations of undifferentiated hESCs (day 0 hEBs) and are expressed as “fold expression from undifferentiated hESC.” Shown above graphs are the corresponding agarose gels of PCR products obtained at linear phases of qPCR reactions. Standard deviations between duplicate or triplicate samples are shown. PECAM indicates platelet-endothelial cell adhesion molecule 1. (C) Developmental progression of hematoendothelial surface marker expression on disaggregated hEB cells at various time points. “% positive hEB cell” represents fluorescence value obtained by FACS analysis following subtraction of control sample background. Each time point represents the mean of 3 to 5 independent experiments with indicated standard deviations.