AIMS/HYPOTHESIS: We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS(+)) cells derived in vitro. Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS(+) cells. Differentiation of INS ( GFP/w ) hESCs using published protocols demonstrated that all GFP(+) cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP(+) cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP(+) cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP(+) cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS(+) cells. CONCLUSIONS/INTERPRETATION: INS ( GFP/w early INS(+) progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS(+) cells from differentiating hESCs.