Understanding molecular mechanisms is key to interpret biological processes to improve human health. Using a stem cell model we have manipulated environmental growth conditions to probe molecular mechanisms under homeostasis. Fully defined ‘2i’ growth conditions induce large scale gene expression changes in pluripotent stem cells versus serum growth conditions. This leads to a dynamic change in DNA methylation which is caused by the Prdm14 upstream transcription regulator. Using single-cell transcription profiling we also discover a subset of genes with dynamic heterogeneous expression in 2i and identify the regulatory mechanisms driving this. These results support the hypothesis that heterogeneity acts as a mechanistic regulator of pluripotency. Lastly, we show the growth environment interacts with male and female sex differences, with metabolic changes arising from both sex and autosomal genes. Our findings emphasise the power of this model system to discover the molecular mechanisms impacting cell function. This enables targeted treatments when homeostasis is disrupted towards effective and specific health outcomes.