Normal development of hematopoietic cells from Tie2^-/- ES cells. (A) FACS analysis of the expression of Flk1 and Tie2 in Tie2^-/- and Tie2^+/- ES cell–differentiated cells. Red squares show the Flk1⁺ fraction. The percentages of cells in each quadrant are indicated. (B) Expression of genes associated with mesoderm in Flk1⁺ cells shown in panel A (red squares) was analyzed by RT-PCR. (C) Flk1⁺ cells (20 000) were cultured on OP9 cells. The number of hematopoietic clusters from Tie2^+/- and Tie2^-/- cells at day 7 of culture was calculated. Results are expressed as the mean ± SD. (D) Tie2^-/- hematopoietic clusters were cultured for an additional 7 days on fresh OP9 cells. The expression of CD45, Mac-1, Gr-1, and Ter119 was analyzed by flow cytometry. ES cell–derived hematopoietic cells are represented by purple shaded histograms; unstained controls, by green lines.