We have developed a miniaturized microfluidic culture system that allows experimentation on individual human embryonic stem cell (hESC) colonies in dynamic (flow applied) or static (without flow) conditions. The system consists of three inlet channels that converge into a cell-culture channel and provides the capability to spatially and temporally deliver specific treatments by using patterned laminar fluid flow to different parts of a single hESC colony. We show that microfluidic culture for 96 h with or without flow results in similar maintenance of hESC self-renewal, the capability to differentiate into three germ cell lineages, and to maintain a normal karyotype, as in standard culture dishes. Localized delivery of a fluorescent nucleic acid dye was achieved with laminar flow, producing staining only in nuclei of exposed cells. Likewise, cells in desired regions of colonies could be removed with enzymatic treatment and collected for analysis. Re-coating the enzyme treated area of the channel with extracellular matrix led to re-growth of hESC colonies into this region. Our study demonstrates the culture of hESCs in a microfluidic device that can deliver specific treatments to desired regions of a single colony. This miniaturized culture system allows in situ treatment and analysis with the ability to obtain cell samples from part of a colony without micromanipulation and to perform sensitive molecular analysis while permitting further growth of the hESC colony.