Maintaining the genetic stability of human pluripotent stem cells (hPSCs) is important for disease modelling and in regenerative medicine. However, hPSCs are shown to acquire non-random chromosomal abnormalities during their growth in culture, conferring the cells with a growth advantage. To identify and quantify the selective pressures experienced by hPSCs in vitro, we co-cultured hPSC lines in pools, in different culture conditions, with samples collected at each passage. Cell lines co-cultured in a pool grow at different rates, resulting in pool imbalance (skew). The proportion of each cell line in the pool can be identified utilising the unique genetic variation within each cell line, obtained from its whole genome sequencing (WGS), whole exome sequencing (WES), database of genotypes and phenotypes (dbGaP) information. We developed computational tools to estimate the unique variants reflecting the abundance of a cell line in the pool. By measuring the rate of skew in stem cell pools, we can screen for conditions that reduce the skew and are associated with reduced selective pressures and fewer cancer-associated mutations. A comprehensive set of culture conditions that include different hPSC culture media, substrates, dissociation methods and supplements were tested to identify the components and their impact on the genomic stability of hPSCs in vitro.