Breakthrough cell therapies for the treatment of cancers require the separation of specific cells, such as T cells, from the patient's blood. Current cell therapy processes rely on magnetic separation, which adds clinical risk and requires elevated manufacturing controls due to the added foreign material that constitutes the magnetic beads. Acoustophoresis, a method that uses ultrasound for cell separation, has demonstrated label-free enrichment of T cells from blood, but residual other lymphocytes limit the ultimate purity of the output T cell product. Here, to increase the specificity of acoustophoresis, we use affinity reagents to conjugate red blood cells with undesired white blood cells, resulting in a cell–cell complex (rosette) of increased acoustic mobility. We achieve up to 99% purity of T cells from blood products, comparable to current standards of magnetic separation, yet without the addition of separation particles.