Although all human ESC (hESC) lines have similar morphology, express key pluripotency markers, and can differentiate toward primitive germ layers in vitro, the lineage-specific developmental potential may vary between individual lines. In the current study, four hESC lines were cultured in the same feeder-free conditions to provide a standardized platform for interline analysis. A high-throughput, forced-aggregation system involving centrifugation of defined numbers of hESCs in V-96 plates (V-96FA) was developed to examine formation, growth, and subsequent cardiomyocyte differentiation from >22,000 EBs. Homogeneity of EBs formed by V-96FA in mouse embryo fibroblast-conditioned medium was significantly improved compared with formation in mass culture (p < .02; Levene's test). V-96FA EB formation was successful in all four lines, although significant differences in EB growth were observed during the first 6 days of differentiation (p = .044 to .001; one-way analysis of variance [ANOVA]). Cardiomyocyte differentiation potential also varied; 9.5% +/- 0.9%, 6.6% +/- 2.4%, 5.2% +/- 3.1%, and 1.6% +/- 1.0% beating EBs were identified for HUES-7, NOTT2, NOTT1, and BG01, respectively (p = .008; one-way ANOVA). Formation of HUES-7 V-96FA EBs in defined medium containing activin A and basic fibroblast growth factor resulted in 23.6% +/- 3.6% beating EBs, representing a 13.1-fold increase relative to mass culture (1.8% +/- 0.7%), consistent with an observed 14.8-fold increase in MYH6 (alphaMHC) expression by real-time polymerase chain reaction. In contrast, no beating areas were derived from NOTT1-EBs and BG01-EBs formed in defined medium. Thus, the V-96FA system highlighted interline variability in EB growth and cardiomyocyte differentiation but, under the test conditions described, identified HUES-7 as a line that can respond to cardiomyogenic stimulation.