Withdrawal of Notch signaling permitted Gr-1⁺/Mac1⁺ ImC differentiation. (A) HPCs were cultured on fibroblasts with either GM-CSF or M-CSF for 7 days. Gr-1⁺ cells were isolated and stained with antibodies against markers of hematopoietic progenitors: APC-conjugated c-Kit, FITC-conjugated Sca-1, and PE-conjugated CD34 and analyzed on a FACSCalibur flow cytometer. (B) HPCs were cultured on fibroblasts with GM-CSF for 5 days; CD45⁺ cells were isolated and further cultured without fibroblasts for additional 5 days with either GM-CSF or M-CSF. In the case of GM-CSF culture, 5 ng/mL TNF-α was added 48 hours before cell phenotype analysis. Cells were labeled with APC-conjugated anti-CD11c or anti–Gr-1 antibodies, PE-conjugated anti-CD11b, and FITC-conjugated anti-IA^q, anti–B7-2, or anti-F4/80 antibodies and analyzed on a FACSCalibur flow cytometer. Values are the average ± SE from 3 experiments. Similar results were obtained when cells were cultured for an additional 5 days on control fibroblasts.