Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. Methods: RNA-seq libraries were prepared using the Illumina TruSeq RNA kit and the TrueSeq method was employed for mRNA enrichment. The libraries were quantified and samples were multiplexed in each lane of the flowcell. Cluster generation was performed and then sequenced on the Illumina HiSeq1000 system. Reads were mapped on the Human Genome Reference and normalized expression table was generated. Results: Among differentially expressed genes 53 of them were up-regulated and 75 were down-regulated. Conclusions: Data demonstrate increased expression of genes associated with growth development and pro-survival in cardiac progenitors cultured under simulated microgravity compared with those cultured under standard gravity. RNA-sequencing analysis was performed to compare global gene expression profiles of cells at differentiation day 8 under simulated microgravity vs. standard gravity.