There are seven linker histone variants in human somatic cells (H1.0 to H1.5 and H1X), and their prevalence varies as a function of cell type and differentiation stage, suggesting that the different variants may have distinct roles. We have revisited this notion by using new methodologies to study pluripotency and differentiation, including the in vitro differentiation of human embryonic stem (ES) and teratocarcinoma cells and the reprogramming of keratinocytes to induced pluripotent stem cells. Our results show that pluripotent cells (PCs) have decreased levels of H1.0 and increased levels of H1.1, H1.3, and H1.5 compared with differentiated cells. PCs have a more diverse repertoire of H1 variants, whereas in differentiated cells, H1.0 expression represents ∼80% of the H1 transcripts. In agreement with their prevalent expression in ES cells, the regulatory regions of H1.3 and H1.5 genes were found to be occupied by pluripotency factors. Moreover, the H1.0 gene promoter contains bivalent domains (H3K4me2 and H3K27me3) in PCs, suggesting that this variant is likely to have an important role during differentiation. Indeed, the knockdown of H1.0 in human ES did not affect self-renewal but impaired differentiation. Accordingly, H1.0 was recruited to the regulatory regions of differentiation and pluripotency genes during differentiation, confirming that this histone variant plays a critical role in the regulation of these genes. Thus, histone H1 variant expression is controlled by a variety of mechanisms that produce distinct but consistent H1 repertoires in pluripotent and differentiated cells that appear critical to maintain the functionality of such cells.