Conditional Pten deletion and ectopic expression of c-Myc allows for survival, differentiation, and proliferation of DN3 cells across the β-selection checkpoint in the absence of Notch signals. Developmental progression of culture-derived Rag2^−/−PTEN^{+/+;Lck−cre+} or Rag2^−/−PTEN^{f/f;Lck−cre+} DN3 cells retrovirally cotransduced to express TCRβ (GFP⁺) and MIY or c-Myc (YFP⁺) and cultured with OP9-Ctrl or OP9-DL1 cells for 6 days. (A) Flow cytometric analysis of CD4 and CD8 cell surface expression is shown for GFP⁺, YFP⁺, CD45⁺ gated cells, whereas panels B and C show the corresponding DP cellularity and fold expansion, respectively, as indicated. Lin⁻ c-Kit⁺ Sca-1⁺ cells sorted from bone marrow of PTEN^{f/f;Lck−cre+} or PTEN^{+/+;Lck−cre+}Rag2^−/− mice were cultured with OP9-DL1 cells for 14 days, retrovirally transduced, then sorted for YFP⁺ and GFP⁺ DN3 cells, and cultured as indicated. Fold expansion was obtained from the total cellularity divided by the number of cells used at the start of the culture (input), and DP cellularity by multiplication of the total cellularity by the percentage of DP cells present in the cultures. Results are representative of 3 independent experiments.