Hhex^−/Δ progenitors show defective lymphoid development in vitro, which can be rescued by overexpression of Ccnd1 or Bcl2. (A) LSK and CLP cells of the indicated Hhex genotypes were cultured in a variety of in vitro culture conditions to stimulate the development of B cells, T cells, DCs, and NK cells. Then, 12 days later, development of B cells (B220⁺CD19⁺) (top), T-cells (Thy1.2⁺CD25⁺) (second from top), DCs (CD11c⁺) (third from top), and NK cells (NK1.1⁺) (bottom) were assessed by flow cytometry. Data are representative of 3 experiments for CLP and 6 experiments for LSK. (B) Graphs showing enumeration of B cells, T cells, DCs, and NK cells obtained from in vitro cultures of progenitors cultured, as in (A), as well as myeloid cells from cells cultured in IL-3 and IL-6 plus stem cell factor. (C) Hex^+/fl and Hhex^−/Δ LSK cells were transduced with the indicated retroviruses and cultured in conditions to support development of B cells (top), T cells (middle), or DCs (bottom). Then, 12 days later, the number of each population was determined. (D) Graphs showing enumeration of B cells (left), T cells (center), and DCs (right) from cultures as in (C). Data are mean ± standard error of the mean of 6 experiments, expressed as a percentage of the MSCV-IRES-GFP (MIG) transduced Hhex^+/fl LSK cultures. FSC, forward scatter. *P < .05; ***P < .001 by Student t test.