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Noninvasive biomarkers hold important potential for the characterization and purification of stem cells because the addition of exogenous labels, probes, or reporters, as well as the disruption of cell-cell and cell-extracellular matrix interactions, can unintentionally but dramatically alter stem cell state. We recently showed that intensity of the intrinsically fluorescent metabolite, nicotinamide adenine dinucleotide (NADH), fluctuates predictably with changes in stem cell viability and differentiation state. Here, we use multiphoton flow cytometry developed in our laboratory to rapidly and noninvasively characterize and purify populations of intact stem cell aggregates based on NADH intensity and assessed the differentiation capacity of sorted populations. We found removal of aggregates with NADH intensity indicative of cell death resulted in a remaining population of aggregates significantly more likely to produce beating cardiomyocytes (26% vs. 8%, P < 0.05). Similarly, we found isolation of stem cell aggregates with NADH intensity indicative of future cardiac differentiation gave rise to more aggregates with beating cardiomyocytes at later time points (50% vs. 28%, P < 0.05). Further, coupling NADH intensity with gating based on size, enhances the enrichment for EBs capable of giving rise to cardiomyocytes (59% vs. 27%, P < 0.05). Thus, we demonstrate that endogenous properties of cell aggregates, such as NADH and size, can serve as gating parameters for large particle sorting devices to purify populations of stem cells or their progeny in a noninvasive manner, leading the way for improved therapeutic applications., (© 2014 International Society for Advancement of Cytometry.)