Acoustophoretic devices and methods for separating biological cells (particularly T-cells) from other fluids/materials using multi-dimensional acoustic standing waves are disclosed. The devices include an inlet, at least two outlets, and a flow chamber having an ultrasonic transducer-reflector pair. Specifically, T cells, B cells, or NK cells can be separated from other blood components. A dual-pass acoustophoretic system including two acoustophoretic devices arranged in series and fluidly connected to one another is also illustrated. Means for pre-chilling the mixture prior to separation in the devices or system can be used to improve retention, concentration, and clarification and to prevent outgassing.