We present a microfluidic platform for simultaneous on-chip pumping and size-based separation of cells and particles without external fluidic control systems required for most existing platforms. The device utilizes an array of acoustically actuated air/liquid interfaces generated using dead-end side channels termed Lateral Cavity Acoustic Transducers (LCATs). The oscillating interfaces generate local streaming flow while the angle of the LCATs relative to the main channel generates a global bulk flow from the inlet to the outlet. The interaction of these two competing velocity fields (i.e. global bulk velocity vs. local streaming velocity) is responsible for the observed separation. It is shown that the separation of 5 μm and 10 μm polystyrene beads is dependent on the ratio of these two competing velocity fields. The experimental and simulation results suggest that particle trajectories based only on Stokes drag force cannot fully explain the separation behavior and that the impact of additional forces due to the oscillating flow field must be considered to determine the trajectory of the beads and ultimately the separation behavior of the device. To demonstrate an application of this separation platform with cellular components, smaller red blood cells (7.5 ± 0.8 μm) are separated from larger K562 cells (16.3 ± 2.0 μm) with viabilities comparable to those of controls based on a trypan blue exclusion assay.