Generation of CD3⁺ T cells from human CD34^hi thymic lymphoid precursors expressing a constitutively active form of Notch1. Development of T cells from human CD34^hi thymic lymphoid precursors transduced with a retroviral vector encoding either the entire intracellular domain of human Notch1 (ICN1) and GFP from a bicistronic transcript, or GFP alone, was analyzed in a hybrid hu/mo FTOC assay. Absolute numbers of total (A) and CD3⁺ cells (B) derived from ICN1-(▪) or mock-transduced (○) progenitors calculated on electronically gated GFP⁺ cells at the indicated culture times. Absolute numbers were normalized to 2 × 10³ GFP⁺ transduced cells/lobe. (C) CD3 expression levels determined by flow cytometry on GFP⁺-gated cells derived from ICN1-transduced (ICN1) or mock-transduced (GFP) progenitors after 32 days of culture (shaded histograms). Background staining (empty histograms) was determined with isotype-matched irrelevant antibodies. Results are representative of 4 independent experiments.