Methods of three-dimensional (3D) cell culture have made significant progress in recent years due to a better understanding of cell to cell interactions and the cell's interface with their surrounding environment. We hypothesized that a microgravity 3D culture system would improve upon the growth and function of a pancreatic progenitor cell population. We developed a rotating wall vessel bioreactor and established a culture system using a pancreatic cell line. Cells in the bioreactors showed robust proliferation, enhanced transcriptional signaling, and improved translation of pancreatic genes compared with two-dimensional static culture. Cells also gained the ability to respond to glucose stimulation, which was not observed in the control cultures. These findings suggest that a 3D microgravity bioreactor environment mimics the niche of the pancreas yielding a cell source with potential for cell-based therapy in the treatment of diabetes.