Formation of hES3/ENVY chimeric EBs enables the frequency of hematopoietic precursors to be estimated. (A) Bright-field (BF) and fluorescence (GFP) images of day 7 chimeric EBs formed from a total of 3000 hESCs consisting of the indicated ratios of GFP^– hES3 and GFP⁺ ENVY cells. Original magnification, 50 ×; objective used was 5 ×/0.12 NA. Further differentiation of the EBs led to the formation of hematopoietic cells in each well, which were all GFP^– (left panels) or all GFP⁺ (right panels) or included a mixture of GFP^– and GFP⁺ cells (middle panels), depending on the proportion of ENVY cells contributing to the EBs. Original magnification, 100 ×; objective used was 10 ×/0.30 NA. Camera and microscope used are as in Figure 1. (B) Plotting the frequency of wells with no GFP⁺ cells against the ENVY input cell number/well demonstrated an excellent correlation (R² > 0.99). Results shown are the mean of 3 experiments with error bars representing the SD. The clonogenic frequency of hematopoietic precursors (1:523) was estimated by determining the ENVY input cell number that resulted in 37% of the wells containing only GFP^– blood cells.