DNA replication initiation is a tightly regulated process critical for genome stability. RECQ4 is essential in this process, and its dysfunction is implicated in genetic disorders such as Rothmund-Thomson syndrome. To better understand RECQ4’s molecular role, we investigated its interactions with replication initiation factors MCM10 and CDC45. We found that MCM10 stimulates RECQ4’s DNA binding and promotes liquid-liquid phase separation—a process thought to help spatially organize replication factors. In contrast, CDC45 inhibits both properties. Despite their opposing effects, all three proteins form a complex. These findings offer new insight into RECQ4 regulation and may help clarify its role in disease.