This study is aimed to enhance the understanding of the interaction between ionic liquids (ILs) and non-ionic Pluronic triblock copolymers in aqueous two-phase micellar systems (ATPMS) used for the selective separation/purification of hydrophobic biomolecules. The ILs allow a precise control of the cloud point phase separation temperature (CPT), particularly important when the stability of the molecule is highly dependent on temperature. The effect of choline-based ILs, with two different counter-anions, chloride and hexanoate, was evaluated using molecular dynamics simulations (MD) for F-68 and L-35 Pluronic aqueous solutions. The simulations revealed the role played by the anions during the Pluronic self-assembly, with choline chloride hindering Pluronic aggregation and the choline hexanoate favouring micelle formation and coalescence, in agreement with the experimental data. A detailed study of the accessible surface area of Pluronic showed a progressive dehydration of the Pluronic hydrophilic micelle corona in choline hexanoate mixtures promoting inter-micelle interactions and, consequently, micelle coalescence. With the addition of choline hexanoate, it was observed that the hydrophilic segments, which form the micelle corona, twisted towards the Pluronic micelle core. The electrostatic interaction is also shown to play a key role in this IL-Pluronic aqueous solution, as the hexanoate anions are accommodated in the Pluronic micelle core, while the choline cations are hosted by the Pluronic micelle corona, with the ions interacting with each other during the self-assembly process. In addition, a comparison study of F-68 and L-35 aqueous solutions shows that the IL impact depends on the length of the Pluronic hydrophilic segment. This work provides a realistic microscopic scenario of the complex interactions between Pluronic copolymers and ILs.