Mast cell progenitors proliferate in the absence of SCF. CD34-enriched cells purified from buffy coats were stained with CellTrace Far Red. (A) The cells were cultured with IL-3 and IL-6 for 5 days and analyzed by flow cytometry. Live singlets were gated. The red-filled histogram represents unstimulated CD34⁺ cells, the blue line indicates CD117^hiFcεRI⁺ pre–mast cells, and the dotted light green line indicates cells that did not fall into the CD117^hiFcεRI⁺ pre–mast cell gate. (B) Cell proliferation was analyzed using the proliferation platform in FlowJo. The model was adjusted to 8 peaks. The non–pre-mast-cell population was used as a positive control for cell division, and the cell generation gates were calculated accordingly. The undivided peak was set according to the unstimulated sample, which was stored at 2°C–8°C throughout the culture period. The division index corresponds to the mean number of cell divisions undergone by the cells in the original culture. The bars represent the means ± SEM of 3 buffy coats. One-way ANOVA with Tukey’s multiple comparisons test. ****Adjusted P < .0001.