AbstractWe report a new aqueous polymerization‐induced self‐assembly (PISA) formulation that enables the hydrophobic block to be prepared first when targeting diblock copolymer nano‐objects. This counter‐intuitive reverse sequence approach uses an ionic reversible addition–fragmentation chain transfer (RAFT) agent for the RAFT aqueous dispersion polymerization of 2‐hydroxypropyl methacrylate (HPMA) to produce charge‐stabilized latex particles. Chain extension using a water‐soluble methacrylic, acrylic or acrylamide comonomer then produces sterically stabilized diblock copolymer nanoparticles in an aqueous one‐pot formulation. In each case, the monomer diffuses into the PHPMA particles, which act as the locus for the polymerization. A remarkable change in morphology occurs as the ≈600 nm latex is converted into much smaller sterically stabilized diblock copolymer nanoparticles, which exhibit thermoresponsive behavior. Such reverse sequence PISA formulations enable the efficient synthesis of new functional diblock copolymer nanoparticles.