Wnt signaling plays a central role in tissue development and homeostasis, and its deregulation is implicated in many human diseases, including cancer. As an essential posttranslational modification, protein phosphorylation is critical in Wnt signaling and has been a focus of investigation using systematic approaches, including proteomics. Typically, studies were conducted by applying purified Wnt ligands to cells in a "starvation" condition to minimize the background noise. Despite leading to many important discoveries, such an approach may omit pivotal integrative effects of Wnt signaling in a complex physiological environment. In this study, we investigated the temporal dynamics of the phosphoproteome following treatments of Wnt3a conditioned medium (CM) with serum supply. This revealed three clusters of phosphoproteome changes with distinct temporal profiles with implications in gene expressions and chromatin organizations. Among these, we observed enhanced phosphorylation at the Thr543 residue of 53BP1, which is a key event in the cellular response to DNA damage. Functionally, it triggered the replication stress response pathway mediated by γH2AX accumulation and Chk1 activation, leading to a significant reduction of cells in the S phase of the cell cycle. Intriguingly, Wnt3a treatment in the serum-free condition did not activate 53BP1-Chk1 and replication stress response. Our study indicates the importance of noting the presence or absence of serum supply when studying the signaling pathways.