We describe methods to isolate cells at successive stages during in vitro differentiation of human stem cells into the pancreatic endocrine lineage. Using flow cytometry, we purify live lineage intermediates in numbers not available by fetal biopsy. These include pancreatic and endocrine progenitors, isolated based on known surface markers. We further report a strategy that leverages intracellular zinc content and DPP4/CD26 expression to separate monohormonal insulin+ β cells from polyhormonal counterparts. These methods enable comprehensive molecular profiling during human islet lineage progression.