It has been some 40 years since repeating subunits in eukaryotic chromatin, initially termed "nu bodies," were described. Four decades of study have characterized the structural organization of the nucleosome, from multiple crystal structures of individual nucleosomes to genome-wide maps of nucleosome positions in scores of organisms. Nucleosome positioning can impact essentially all DNA-templated processes, making an appreciation of the forces shaping the nucleosomal landscape in eukaryotes key to fully understanding genomic regulation. Here, we review the factors impacting nucleosome positioning and the ways that nucleosomes can control the output of the genome.