Ultrasonic manipulation has emerged as a simple and powerful tool for trapping, aggregation, and separation of cells. During the last decade, an increasing amount of applications in the microscale format has been demonstrated, of which the most important is acoustophoresis (continuous acoustic cell or particle separation). Traditionally, the technology has proven to be suitable for treatment of high-density cell and particle suspensions, where large cell and particle numbers are handled simultaneously. In this chapter, we describe how ultrasound can be combined with microfluidics and microplates for particle and cell manipulation approaching the single-cell level. We demonstrate different cell handling methods with the purpose to select, trap, aggregate, and position individual cells in microdevices based on multifrequency ultrasonic actuation, and we discuss applications of the technology involving immune cell interaction studies.