DNA replication, the duplication of genetic and epigenetic information, is the basis of biological inheritance. In all eukaryotes, DNA replication follows a tightly regulated replication-timing program, which dynamically changes during development and has a lot of conserved characteristics across multiple species. However the molecular mechanism of the replication timing program remains elusive. Using a Xenopus egg extract in vitro replication system, our lab found that the replication timing program is established at a discrete window of time during early G1-phase, termed the Timing Decision Point (TDP). The TDP is coincident with the re-positioning and anchorage of chromosome segments in the newly formed nucleus. Previous studies have found that many chromatin related factors were not able to determine replication timing. Here, I discovered that neither the non-coding satellite RNAs from mouse pericentric heterochromatin nor histone methyltransferase G9a was the main replication timing determinant. I further examined the replication timing property of chromatin in G2-phase and quiescent (G0) cells and demonstrated that G2-phase chromatin lacks determinants of replication timing but maintains the overall spatial organization of chromatin domains, using both in vitro system and in vivo cell culture. 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 establish chromatin marks as replication timing determinants, potentially facilitated by spatial organization, which persist independent of such organization until the process of chromatin replication dilutes or disrupts those determinants. These results demonstrate that establishment and maintenance of the replication timing program are mediated by different mechanisms, both of which potentially involve chromatin spatial organization. This work represents a major advance in our quest for the replication timing determinants and understanding of the mechanism of the replication timing program.