PML nuclear body (NB) malfunction often leads to acute leukemia outbreaks and other severe diseases. PML NB rescue is the molecular basis of arsenic success in acute promyelocytic leukemia (APL) treatment. However, it is unclear how PML NBs are assembled. Here, we observed the presence of liquid–liquid phase separation (LLPS) in NB formation by fluorescence recovery after photobleaching (FRAP) experiment. Compared with the wild‐type (WT) NBs, PML A216V derived from arsenic‐resistant leukemia patients markedly crippled LLPS, but not altered the overall structure and PML RBCC oligomerization. In parallel, we also reported several Leu to Pro mutations that were critical to PML coiled‐coil domain. FRAP characterization and comparison between L268P and A216V revealed markedly different LLPS activities in these mutant NBs. Transmission electron microscopy (TEM) inspections of LLPS‐crippled and uncrippled NBs showed aggregation‐ and ring‐like PML packing in A216V and WT/L268P NBs, respectively. More importantly, the correct LLPS‐driven NB formation was the prerequisite for partner recruitment, post‐translational modifications (PTMs), and PML‐driven cellular regulations, such as ROS stress control, mitochondria production, and PML‐p53‐mediated senescence and apoptosis. Altogether, our results helped to define a critical LLPS step in PML NB biogenesis. Working hypothesis of LLPS‐driven PML NB biogenesis. In the first step, PML–PML interaction may be mainly mediated by RBCC oligomerization. In the second step, PML multimers can come together through LLPS, facilitated by local protein concentrations, PTMs, and recruitment of partner proteins, ultimately giving rise to a remarkable subcellular organelle with flexible sizes.