Generation of functional β cells from pluripotent sources would accelerate diagnostic and therapeutic applications for diabetes research and therapy. However， it has been challenging to generate competent β cells with dynamic insulin-secretory capacity to glucose and incretin stimulations. We introduced transcription factors， critical for β-cell development and function， in differentiating human induced pluripotent stem cells (PSCs) and assessed the impact on the functionality of derived β-cell (psBC) progeny. A perifusion system revealed stepwise transduction of the PDX1， NEUROG3， and MAFA triad (PNM) enabled in?vitro generation of psBCs with glucose and GLP-1 responsiveness within 3?weeks. PNM transduction upregulated genes associated with glucose sensing， insulin secretion， and β-cell maturation. In recipient diabetic mice， PNM-transduced psBCs showed glucose-responsive insulin secretion as early as 1?week post transplantation. Thus， enhanced pre-emptive β-cell specification of PSCs by PNM drives generation of glucose- and incretin-responsive psBCs in?vitro， offering a competent tissue-primed biotherapy.