Cellular separations are required in many contexts in biochemical and biomedical applications for the identification, isolation, and analysis of phenotypes or samples of interest. Microfluidics is uniquely suited for handling biological samples, and emerging technologies have become increasingly accessible tools for researchers and clinicians. Here, we review advances in the last few years in techniques for microfluidic cell separation and manipulation. Applications such as high-throughput cell and organism phenotypic screening, purification of heterogeneous stem cell populations, separation of blood components, and isolation of rare cells in patients highlight some of the areas in which these technologies show great potential. Continued advances in separation mechanisms and understanding of cellular systems will yield further improvements in the throughput, resolution, and robustness of techniques.