AbstractRapid self‐renewal of human embryonic stem (ES) cells (NIH designation WA01 and WA09) is accommodated by an abbreviated cell cycle due to a reduction in the G1 phase. Thus, molecular mechanisms operative in ES cells may expedite the cellular commitment to progress into S phase to initiate replication of DNA and biosynthesis of histone proteins to form new chromatin. Here we show that the selective cell cycle regulated expression of individual histone H4 gene copies, which is typical for somatic cell types, is already firmly established in human ES cells. This early establishment of H4 gene regulation, which is E2F independent, is consistent with co‐expression of the cognate transcriptional regulators HiNF‐P and p220NPAT. Human ES cells differ from somatic cells in the expression of members of the E2F family and RB‐related pocket proteins (p105RB1, p107RBL1, and p130RBL2/RB2) that control expression of genes encoding enzymes for nucleotide metabolism and DNA synthesis. Human ES cells rapidly and robustly (>200‐fold) induce the cyclin dependent kinase (CDK) inhibitor p21WAF1/CIP1 upon gamma‐irradiation. This DNA damage response promptly reduces histone gene expression as well as mRNA levels for HiNF‐P and p220NPAT and causes accumulation of unprocessed histone H4 precursor RNAs. Furthermore, while E2F4, E2F5 and p130RBL2/RB2 are the major E2F and pocket protein mRNAs in actively proliferating ES cells, expression levels of E2F5, E2F6, and p105RB1 are most strongly elevated during cell cycle arrest in cells responding to DNA damage. Our data suggest that the brief G1 phase of ES cells is supported by a potent p21WAF1/CIP1 related DNA damage response that functions through several mechanisms to rapidly inhibit cell cycle progression. This response may alter the E2F/pocket protein combinations that control E2F dependent genes and block H4 gene expression by inhibiting histone‐specific transcription factors and processing of histone gene transcripts, as well as by destabilizing histone mRNAs. J. Cell. Physiol. 210: 517–526, 2007. © 2006 Wiley‐Liss, Inc.