Human induced pluripotent stem cells (hiPSCs) provide a potential resource for regenerative medicine. To identify the signalling pathway(s) contributing to the development of functional β cells, we established a tracing model consisting of dual knock-in hiPSCs (INS-Venus/NGN3-mCherry) (hIveNry) expressing the fluorescent proteins Venus and mCherry under the control of intrinsic insulin (INS) and neurogenin 3 (NGN3) promoters, respectively. hIveNry iPSCs differentiated into NGN3- and mCherry-positive endocrine progenitors and then into Venus-positive β cells expressing INS, PDX1, NKX6.1, and glucokinase (GCK). Using these cells, we conducted high-throughput screening of chemicals and identified a specific kinase inhibitor of fibroblast growth factor receptor 1 (FGFR1) that acted in a stage-dependent manner to promote the terminal differentiation of pancreatic endocrine cells, including β cells, from the intermediate stage of pancreatic endocrine progenitors while blocking the early development of pancreatic progenitors. This FGFR1 inhibitor augmented the expression of functional β cell markers (SLC30A8 and ABCC8) and improved glucose-stimulated INS secretion. Our findings indicate that the hIveNry model could provide further insights into the mechanisms of hiPS-derived β cell differentiation controlled by FGFR1-mediated regulatory pathways in a temporal-dependent fashion.