An experimental study of water flow in a T-shaped channel with rectangular cross section (20×20 mm2 inlet ID and 20×40 mm2 outlet ID) has been conducted for a Reynolds number Re range based on inlet geometry of Re = 56–422. Dynamical conditions and T-channel geometry of the current study are applicable to the microscale. This study supports a large body of numerical work, and resolution and the interrogation region are extended beyond previous experimental studies. Laser induced fluorescence (LIF) permits a detailed look at the flow fields that evolve in the outlet channel over the broad range of Re where realistic T-channels operate. Scalar structures previously unresolved in the literature are presented. Unsteady flow regimes numerically predicted to occur at higher Re are characterized, and simultaneous planar and discrete-point LIF measurements relate the development of oscillatory behavior in the outlet channel to flow structure in the junction. Further, the development of an unsteady symmetric topology at higher Re, which negatively affects mixing, is presented, and mechanisms behind the wide range of mixing qualities predicted for this regime are explained. Characteristics of steady and unsteady flows are tracked with Re to elucidate mixing behavior on a fundamental level. Practical conclusions for experimental mixing in a T-channel are extracted.