Flow field-flow fractionation (FlFFF) is a liquid-phase separation technique that can separate macromolecules and particles on the basis of differences in their diffusion coefficient, and therefore their size. The membrane in the FlFFF channel is one of the most important elements involved in accomplishing the separation. This mini-review focuses on requirements and important points related to membranes used in FlFFF channels. Unless it is selected properly, the membrane can be a weak point of the technique due to risks such as sample loss because of the interactions between the sample and the membrane.