In a previous study we established colony assays suitable for studying murine adult (2-4 months) pancreatic progenitor cells plated in semisolid media containing methylcellulose and extracellular matrix proteins. Using these assays, we found robust in vitro progenitor cell activities (multilineage differentiation and self-renewal) from pancreatic cells of adult mice in the CD-1 outbred background. However, it was not clear whether progenitor cell activities can be detected from inbred mice, a preferred mouse model for various genetic studies. It was also not clear whether a single cell is sufficient to self-renew. Here, we show that fluorescent activated cell sorting pancreatic CD133(+) but not CD133(-) cells from adult C57Bl/6 inbred mice are enriched for progenitor cells that self-renew and give rise to multilineage colonies in vitro. The number of cells in a colony is in proportion to its diameter. Around 60% of single handpicked 3-week-old colonies express trilineage markers, indicating most progenitors are tripotent for ductal, acinar, and endocrine lineage differentiation. Approximately 80% of primary (freshly sorted) colony-forming progenitor cells are capable of giving rise to secondary progenitors in vitro, indicating that a majority of the primary progenitors self-renew. A single cell is sufficient for self-renewal and a Wnt agonist, R-Spondin1, enhances the number of secondary progenitors from the primary progenitors. Together, our pancreatic colony assays allow quantitative analyses of progenitors at a single-cell level from inbred mice. These assays will be useful for elucidating in vitro mechanisms of pancreatic progenitor cell biology.