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DNA replication in all eukaryotes follows a defined replication timing program, the molecular mechanism of which remains elusive. Using a Xenopus laevis egg extract replication system, we previously demonstrated that replication timing is established during early G1 phase of the cell cycle (timing decision point [TDP]), which is coincident with the repositioning and anchorage of chromatin in the newly formed nucleus. In this study, we use this same system to show that G2 phase chromatin lacks determinants of replication timing but maintains the overall spatial organization of chromatin domains, and we confirm this finding by genome-wide analysis of rereplication in vivo. In contrast, chromatin from quiescent cells retains replication timing but exhibits disrupted spatial organization. These data support a model in which events at the TDP, facilitated by chromatin spatial organization, establish determinants of replication timing that persist independent of spatial organization until the process of chromatin replication during S phase erases those determinants.