Objective: Processing of peripheral blood progenitor cells (PBPC) for clinical transplantation or research applications aims to effectively select or deplete specific cell populations. Usually, fluorescence- or magnetically- based sorting techniques are used for PBPC processing. Here, we aimed to investigate whether a novel microchip- based ´acoustophoresis´ technique can be used for ultrasound separation of cell subsets from PBPC. In acoustophoresis, ultrasonic standing wave forces are applied to sort cells or particles in a continuously flowing suspension in a micro channel. Cells are separated from the surrounding media depending on their physical properties and sorting is primarily based on size, density and compressibility. Materials and Methods: PBPC were obtained from patients and healthy donors undergoing PBPC apheresis. Cells were labeled with Dynal beads and sorted acoustically using a 2D-2Y chip and, in parallel, also magnetically. Sorted cells were analyzed by FACS and standard functional tests. Results: We have previously reported that platelets can be effectively removed from PBPC samples with a depletion rate of about 90% in a label-free sorting system. Based on these data we now went on to investigate whether acoustophoresis could also be applied to realize sorting of defined leucocyte populations. PBPC samples from patients (n= 16) and healthy donors (n=6) were labelled with anti-CD4 microbeads (Dynal) and sorted on an acoustophoresis-microchip. In parallel, control magnetic sorting was performed. Mean purity of targeted CD4+ cells was 87 ± 12% (± SD) for acoustophoresis, compared to 95 ± 7% for control magnetic sorting. Viability of sorted cells was very good for both sorting methods (95 ± 4% and 97 ± 3%, respectively). Acoustophoresis recovery rate of CD4+ cells was 69 ± 19%, compared to 59 ± 19% for magnetic sorting. Furthermore, functional testing of targeted CD4+ cells demonstrated unimpaired mitogen-mediated proliferation capacity and cytokine production. Conclusion: In summary, the acoustophoresis technique can be utilized to efficiently sort bead-labelled lymphocyte populations from PBPC samples with high purity and recovery without impairing lymphocyte function. Acoustophoresis is, thus, an interesting technology for PBPC processing, which has furthermore the potential to offer a single platform technique for multi-parameter cell separation.