In 1966, Pohl and Hawk Science 152, 647–649 (1966) published the first demonstration of dielectrophoresis of living and dead yeast cells; their paper described how the different ways in which the cells responded to an applied nonuniform electric field could form the basis of a cell separation method. Fifty years later, the field of dielectrophoretic (DEP) cell separation has expanded, with myriad demonstrations of its ability to sort cells on the basis of differences in electrical properties without the need for chemical labelling. As DEP separation enters its second half-century, new approaches are being found to move the technique from laboratory prototypes to functional commercial devices; to gain widespread acceptance beyond the DEP community, it will be necessary to develop ways of separating cells with throughputs, purities, and cell recovery comparable to gold-standard techniques in life sciences, such as fluorescence- and magnetically activated cell sorting. In this paper, the history of DEP separation is charted, from a description of the work leading up to the first paper, to the current dual approaches of electrode-based and electrodeless DEP separation, and the path to future acceptance outside the DEP mainstream is considered.