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This study explores a facile synthesis method by simply changing the type of precursor cations or anions to regulate the nanosheet stack size of mesoporous 2D MFI zeolites while preserving the ‘self-interlocked ordered nanosheet stack’ (SI-ONS) microstructural characteristics. The important roles of precursor cations and anions in controlling the structure and hence chemical properties of the 2D MFI zeolites are demonstrated and the corresponding mechanisms are explained. The ability to facilitate microporous MFI framework development followed the order Na+ < K+ < Rb+ < Cs+ for cations and SO4 2− ≈ NO3 − ≈ Cl− for anions. Conversely, the capability to generate inter-crystalline mesopores followed the order Na+ > K+ > Rb+ > Cs+ and SO4 2− > NO3 − > Cl−. The cations and anions showed little effect on the number and strength of strong acid sites, but clearly influenced their hydrothermal stability. The hydrothermal stability of strong acid sites in the resulting 2D MFI zeolites followed the order Na+ < K+ < Rb+ for cations and SO4 2− < Cl− < NO3 − for anions. A general micelle-aggregation mechanism governing the microstructure formation of SI-ONS in precursors containing different types of cations and anions was proposed established. It was found, for the first time, that a linear relationship exists between nanosheet stack size and mesopore volume of 2D MFI zeolites, which provides a valuable indicator for predicting mesopore volume from nanosheet stack size for a 2D MFI zeolite product. Catalytic performance of the synthesized 2D MFI zeolites was examined using three selected liquid phase reactions including the O-methylation of cyclohexanone, the esterification of benzyl alcohol with hexanoic acid and the protection of benzaldehyde with pentaerythritol. It was shown that the catalytic activity of the 2D MFI zeolites was primarily dictated by the amount of mesopores present, for example, the reaction conversion increased by as much as 300% when the mesopore volume increased from 0.05 to 0.55 cm3 g−1.