CRISPR-Cas9 can be used to generate edited cell pools for a variety of applications. These pools contain a heterogeneous mix of cells, some unedited and some with different genetic alterations at the targeted locus. In order to produce a cell line with a particular edit or knockout, clonal expansion of individual cells is necessary. This protocol describes how to isolate single cells from a CRISPR-edited pool, and expand and genotype the subsequent clonal populations. The isolation of single cells from a knockout cell pool can be accomplished through limiting dilution. While there are more sophisticated means to accomplish single cell plating (e.g., flow cytometry), limiting dilution is a technique that can be carried out with standard pipetting tools. The obvious drawback to this technique is that it relies on statistical probability (based on a Poisson distribution) to deposit a single cell per well. A typical concentration used for limiting dilution is 0.5-1 cells per 100 μl aliquot because it maximizes the probability of obtaining a single cell per well while minimizing the probability of depositing multiple cells per well. At this concentration, one can expect about a third of the wells to contain a single cell and most wells to be empty. It is much less difficult to exclude wells without cells than to sort out heterogeneous populations arising from multiple cells. In this protocol, cells from each edited population are diluted to 0.5-1 cells per 100 μl and plated on at least two 96-well plates (100 μl/well). However, if clonally expanding cells for the first time we recommend plating a higher number of cells (e.g., 4 x 96-well plates per condition). To control for potential health/growth defects caused by the editing, we recommend including mock and/or negative control for single cell dilution, although these do not require multiple 96-well plates. The RELA positive control, however, does not need to be plated out for clonal expansion as it serves mainly as a positive control for transfection and editing efficiency.