This paper presents a theoretical and experimental investigation into the hydrodynamic focusing effect in rectangular microchannels. Two theoretical models for two-dimensional hydrodynamic focusing are proposed. The first model predicts the width of the focused stream in symmetric hydrodynamic focusing in microchannels of various aspect ratios. The second model predicts the location and the width of the focused stream in asymmetric hydrodynamic focusing in microchannels with a low or high aspect ratio. In both models, the theoretical results are shown to be in good agreement with the experimental data. Hence, the models provide a useful means of performing a theoretical analysis of flow control in microfluidic devices using hydrodynamic focusing effects. The ability of the proposed models to control the focused stream within a micro flow cytometer is verified in a series of experimental trials performed using polystyrene microparticles with a diameter of 20 µm. The experimental data show that the width of the focused stream can be reduced to the same order of magnitude as that of the particle size. Furthermore, it is shown that the microparticles can be successfully hydrodynamically focused and switched to the desired outlet port of the cytometer. Hence, the models presented in this study provide sufficient control to support cell/particle counting and sorting applications.