To realize the full scientific and clinical potential of human embryonic stem cell (hESC)- derived cardiomyocytes, strategies are required to improve differentiation and overcome the high degree of heterogeneity of differentiated populations. To address these issues, we developed a novel method to force hESCs into three dimensional differentiating aggregates (embryoid bodies) that are of greater homogeneity, and found that this improved the efficiency of cardiomyocyte differentiation. Moreover, cardiomyocytes were enriched via two transgenic approaches: 1) negative selection of proliferating cells with the Herpes Simplex Virus thymidine kinase / ganciclovir (HSVtk / GCV) suicide gene system; and 2) positive selection of cardiomyocytes expressing a bicistronic reporter (green fluorescent protein – internal ribosome entry site – puromycin-N-acetyltransferase) from a cardiac specific promoter. Parental and transgenic cells were similar with regards to morphology, pluripotency marker expression, differentiation potential and cardiomyocyte electrophysiology. Crucially, while immunostaining of dissociated cardiomyocyte preparations expressing HSVtk or puromycin-N- acetyltransferase contained <7% cardiomyocytes, parallel cultures treated with GCV or puromycin contained respectively 33.4±2.1% and 91.5±4.3% cardiomyocytes, corresponding to an enrichment factor of 6.7- and 14.5-fold. Drug-selected cardiomyocytes responded to chronotropic stimulation and displayed cardiac-specific action potentials, demonstrating retention of functionality. Both the aggregation and the transgenic strategies will be generically applicable and should readily translate to the enrichment of many other differentiated lineages derived from hESCs.