Long-term, time-lapse imaging studies of embryonic stem cells (ESCs) require a controlled and stable culturing environment for high-resolution imaging. Microfluidics is well-suited for such studies, especially when the media composition needs to be rapidly and accurately altered without disrupting the imaging. Current studies in plates, which can only add molecules at the start of an experiment without any information on the levels of endogenous signaling before the exposure, are incompatible with continuous high-resolution imaging and cell-tracking. Here, we present a custom designed, fully automated microfluidic chip to overcome these challenges. A unique feature of our chip includes three-dimensional ports that can connect completely sealed on-chip valves for fluid control to individually addressable cell culture chambers with thin glass bottoms for high-resolution imaging. We developed a robust protocol for on-chip culturing of mouse ESCs for minimum of 3 days, to carry out experiments reliably and repeatedly. The on-chip ESC growth rate was similar to that on standard culture plates with same initial cell density. We tested the chips for high-resolution, time-lapse imaging of a sensitive reporter of ESC lineage priming, Nanog-GFP, and HHex-Venus with an H2B-mCherry nuclear marker for cell-tracking. Two color imaging of cells was possible over a 24-hr period while maintaining cell viability. Importantly, changing the media did not affect our ability to track individual cells. This system now enables long-term fluorescence imaging studies in a reliable and automated manner in a fully controlled microenvironment.