Use of RTOCs to generate T cells from ES cell–derived Flk1⁺ progenitors. (A) Cells were harvested from day 5 ES/OP9 coculture, and CD45^– Flk1⁺ progenitors were enriched by magnetic-assisted cell sorting to about 97% purity from about 33% of the starting population. (B) Flk1⁺ cells were combined with a thymic stromal cell suspension and deposited onto FTOC rafts (see “Materials and methods”). After 19 days, RTOC reconstitution was determined by flow cytometric analysis for CD45.2 surface expression. ES cell–derived CD45.2⁺ thymocytes were present in all RTOCs from 5 independent experiments. Flow cytometric analysis is also shown for CD4 and CD8 and for CD3 and TCRβ surface expression on CD45.2⁺ gated cells, with individual reaggregates pooled for analysis. (C) CD117⁺CD24^loCD45.2⁺ fetal liver cells from day 14 fetal mice were combined with thymic stromal cells and placed in RTOCs and cultured for 12 days prior to flow cytometric analysis for CD45.2, CD4 and CD8, and CD3 and TCRβ surface expression.