Primary Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ blood cells constitute mast cell progenitors. (A) Enriched mononuclear cells from blood were analyzed with flow cytometry. (B-C) Lin⁻ CD34^hi CD117^int/hi FcεRI⁻ and Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ cells were cultured in a myeloerythroid cytokine cocktail containing IL-3, IL-5, IL-6, IL-9, IL-11, FMS-like tyrosine kinase 3 ligand, stem cell factor, thrombopoietin, erythropoietin, and granulocyte macrophage–colony-stimulating factor for 7 days and analyzed with flow cytometry and May-Grünwald Giemsa staining. The results in panels B and C are representative of 3 independent experiments. The width of each photo corresponds to 50 μm. (D-E) Lin⁻ CD34^hi CD117^int/hi FcεRI⁻ and Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ cells were cultured for a total of 17 to 19 days in a myeloerythroid cytokine cocktail, and enzyme histochemical staining of tryptase activity was performed to identify tryptase-containing cells. Red/brown color indicates positive staining. The results in panels D and E are representative of 3 independent experiments. The width of the photo in panel D corresponds to 160 μm, whereas the width of each photo in panel E corresponds to 40 μm. (F-G,I) Lin⁻ CD34^hi CD117^int/hi cells were analyzed with flow cytometry. Lin⁻ CD34^hi CD117^int/hi FcεRI⁻ CD45RA⁻ CRTH2⁻ integrin β7⁻ (H) and Lin⁻ CD34^hi CD117^int/hi FcεRI⁺ CD45RA⁻ CRTH2⁻ integrin β7⁺ (J) cells were sorted and cultured in a myeloerythroid cytokine cocktail for 7 days and analyzed with flow cytometry. The results in panels H and J are from 1 out of 2 independent experiments. The frequency of CD117⁺ cells (K) or CD117⁺ FcεRI⁺ cells (L) was analyzed after culturing subpopulations of Lin⁻ CD34^hi CD117^int/hi blood progenitors in a myeloerythroid cytokine cocktail for 7 days. The median values are shown. The differences between groups were evaluated using the 2-tailed Mann-Whitney U test. **P < .01.