It has been exactly 50 years since it was discovered that duplication of the eukaryotic genome follows a defined temporal order as cells progress through S‑phase. While the mechanism of this replication‑timing program still remains a mystery, various correlations of this program with both static and dynamic properties of chromatin render it an attractive forum to explore previously impenetrable higher‑order organization of chromosomes. Indeed, studies of DNA replication have provided a simple and straightforward approach to address physical organization of the genome, both along the length of the chromosome as well as in the context of the 3‑dimensional space in the cell nucleus. In this chapter, we summarize the 50‑years history of the pursuit for understanding the replication‑timing program and its developmental regulation, primarily in mammalian cells. We begin with the discovery of the replication‑timing program, discuss developmental regulation of this program during X‑inactivation in females as well as on autosomes and then describe the recent findings from genome‑wide dissection of this program, with special reference to what takes place during mouse embryonic stem cell differentiation. We make an attempt to interpret what these findings might represent and discuss their potential relevance to embryonic development. In doing so, we revive an old concept of “autosomal Lyonization” to describe “facultative heterochromatinization” and irreversible silencing of individual replication domains on autosomes reminiscent of the stable silencing of the inactive X chromosome, which takes place at a stage equivalent to the postimplantation epiblast in mice.