The spleen is an important organ of the immune system responsible for filtering blood and initiating immune responses to blood-borne antigens. Various blood, lymphoid, and hematopoietic cell types may be isolated from spleen samples for further study of the immune system. Preparing a true single cell suspension of the primary tissue sample will optimize cell separation by avoiding additional cell loss and enabling maximum labeling of the target cells. This protocol describes how to harvest cells from a spleen sample, and prepare a single cell suspension prior to performing cell isolation.