The cells of the blastocyst inner cell mass only remain pluripotent for a short time. This state of pluripotency and self-renewal can be maintained in embryonic stem (ES) cell culture by adding specific chemicals or growth factors to ensure the cells can continue dividing and later differentiate into different cell types. In some cases, the cells are grown on a feeder layer of differentiated cells, which provides the growth factors and extracellular matrix components necessary for stem cell proliferation.

The ES cell state is regulated by the transcription factors, Oct4, Sox2, and Nanog. These factors activate the genes required for maintaining pluripotency and repress those involved in differentiation. Silencing any of these transcription factors results in lineage-specific differentiation. The factors operate along with many other transcription factors, including Klf4, Klf5, and Smad1, and transcriptional cofactors, such as p300, Mediator, and Nipb, help activate or repress genes without directly binding to DNA.

Chromatin regulators play a crucial role in maintaining the ES cell state. These are categorized as histone-modifying enzymes and ATP-dependent chromatin regulators. Histone modifying enzymes alter the DNA-histone interaction and transcriptionally activate specific genes. These enzymes also suppress development regulators such as polycomb group (PCG) protein complexes. ATP-dependent chromatin remodeling complexes use the energy released by ATP hydrolysis to either displace histones from DNA or enable the relocation of histones. This weakens the bonds between histones and DNA, providing access to transcription factors to bind to DNA.