The OP9:OP9DL1 culture system supports development of multiple myeloid lineages from MPP, CLP, and DN1. (A) A total of 100 MPP, CLP, or DN1 cells were sorted into 24-well plates containing a confluent layer of a 1:1 mixture of OP9:OP9DL1 stroma in the presence of Flt3L, SCF, IL-3, IL-6, M-CSF, GM-CSF, G-CSF, and IL-7. After 11 days, wells were analyzed by flow cytometry. Upper plots are gated on donor-derived cells and show gates for Mac-1⁺CD25⁻ myeloid cells and CD25⁺Mac-1⁻ T cells; lower plots are gated on the donor-derived, Mac-1⁺CD25⁻ cells from the upper plots, revealing 3 subpopulations of myeloid cells generated from all 3 sorted progenitors. (B) The Gr-1⁺MHCII⁻, Gr-1⁻MHCII⁻, and MHCII⁺ myeloid subsets shown in panel A were sorted, plated onto slides using a cytospin, and analyzed morphologically by Hemacolor stains.