Pancreatic β cell replacement therapy is considered as a potential strategy to treat diabetes. To date, transplantation of pancreatic islets from cadavers is the most effective approach for treating diabetic patients, but this approach has limitations in terms of the necessity of strong immunosuppressive drugs and the shortage of donors. Alternatively, human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and hiPSCs, can be differentiated into insulin-secreting β cells that have a great potential to treat diabetes. However, generation of functional β cells similar to those present naturally in the pancreas is, so far, a work in progress. Indeed, few studies reported the generation of functional β cells from hPSCs, but those cells were low in efficiency and functionality, creating a major obstacle to the use of these cells for cellular therapy. Recent progress showed that patients with diabetes could be transplanted with hPSC-derived pancreatic progenitors co-expressing the two transcription factors (TFs), PDX1 and NKX6.1. Those progenitors are known as the precursors of functional pancreatic β cells.; therefore, after their transplantation, they are converted into functional insulin-secreting β cells inside patient’s body. Recently, we have established an efficient protocol for maximizing generation of PDX1+/NKX6.1+ pancreatic progenitors from hPSCs. We enhanced the generation of PDX1+/NKX6.1+ population, by manipulating in vitro culture conditions. Our optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1+/NKX6.1+ progenitors (~90%) and upregulation of the key TFs controlling pancreatic β cell development. The improved efficiency of pancreatic differentiation was associated with an inhibition in the hepatic lineage and an increase in the proliferation of NKX6.1+ cells. Further differentiation validated the ability of PDX1+/NKX6.1+ progenitors to generate endocrine progenitors (NGN3+ cells). In addition, we were also able to enrich a novel PDX1-/NKX6.1+ population by manipulating the re-plating densities, that appeared in the form of three-dimensional clusters. Our findings provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1+/NKX6.1+ pancreatic progenitors with an increased self-replicating capacity, thereby aiding scalable production of functional β cells from hPSCs in vitro. Our innovative method also supports the presence of two distinct NKX6.1+ pancreatic populations during human pancreatic development, which may suggest a new trajectory to generate β cells in humans. In this talk, I will present our recently published work on generating distinct pancreatic progenitor populations from hPSCs.