When mouse marrow and spleen cells were cultured for over 12 days in methylcellulose containing media conditioned by pokeweed-mitogen-stimulated spleen cells, colonies containing mast cells and blast cells were observed. The characteristic morphology of the colonies and the time course of their development allowed in situ identification of the mast cell colonies. Identification of the mast cells was confirmed by metachromatic staining with toluidine blue and alcian blue, transmission electron microscopy, and by demonstration of the membrane receptors for IgE. Coculture studies with male and female marrow cells strongly indicated the single cell origin of individual colonies. Detailed cytologic analyses of mixed hemopoietic colonies and replating experiments of individual mixed hemopoietic and mast cell colonies clearly established the hemopoietic origin of mast cells. In replating experiments of individual mast cell colonies, those without blast cells did not yield secondary mast cell colonies. This result strongly indicated that morphologically recognizable mast cells have lost their self-renewing capabilities. The quantitative nature of the mast cell colony assay was supported by linearity studies and provides a method for studies of the progenitors of mouse mast cells.