ADP-ribosylation is an evolutionarily conserved complex posttranslational modification that alters protein function and/or interaction. Intracellularly, it is mainly catalyzed by diphtheria toxin-like ADP-ribosyltransferases (ARTDs), which attach one or several ADP-ribose residues onto target proteins. Several specific mono- and poly-ADP-ribosylation binding modules exist; hydrolases reverse the modification. The best-characterized ARTD family member, ARTD1, regulates various DNA-associated processes. Here, we focus on the role of ARTD1-mediated chromatin ADP-ribosylation in development, differentiation, and pluripotency, and the recent development of new methodologies that will enable more insight into these processes.