Human induced pluripotent stem cells (iPSCs) can differentiate in vitro into insulin-producing cells. The role of cellular age in β-cell differentiation and iPSC-derived β-cell function is unknown. Recent studies reveal that aging leads to an increase in senescent β cells, impairing glucose sensing and insulin secretion. This study aims at investigating aging hallmarks at various differentiation stages and on β-cell function. Six clones of iPSC cell line DRI2 were differentiated into mature β cells (iβ). Aging hallmarks were analysed by measuring the senescence-associated β-Galactosidase activity (SA-β-Gal), the phosphorylation of the histone H2AX (γH2AX) and the superoxide production (mitoSOX) in flow cytometry and immunofluorescence. Genes related to the senescence-associated secretory phenotype (SASP) and cellular senescence were quantified by TaqMan custom gene arrays. The senescence state of the iβ was associated to their function, assessed by dynamic Glucose-Stimulated Insulin Secretion (GSIS). The conditioned media of iβ was tested on EndoC-βH5 cells to evaluate the SASP effects on them. Data show that cell aging increases during differentiation. In fact, flow cytometry showed higher positive cell percentages at the iβ stage compared to iPSCs for SA-β-Gal (iPSC: 9.6%±0.4%; iβ: 42.1%±1.4%), γH2AX (iPSC: 3.5±0.5%; iβ: 18%±0.9%), and mitoSOX (iPSC: 5.6%±0.6%; iβ: 25.4%±4%). Immunofluorescence confirmed γH2AX, SA-β-Gal, and superoxide presence at the iβ stage. Transcriptomic analysis showed increased expression of genes related to SASP, cell cycle arrest, DNA damage, and β-cell senescence in the final maturation stage (iβ). GSIS data showed that cell aging seems to impact on β-cell function. These results suggest that the senescence state of iβ cells may affect differentiation efficiency and β-cell function. Protection from the aging process during differentiation or depletion of senescent cells before transplantation is required for an efficient and long-lasting function of stem cell-derived β cells for cell therapy.