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Embryonic stem cells (ESCs) hold promise either as an in vitro model recapitulating early embryonic development or as a renewable source of therapeutically useful cells. However, 2D culture system, which is still generally utilized in ESCs differentiation strategies, has greatly impeded our learning more information about ESCs proliferation/differentiation in situ. Here, a high aspect ratio vessel (HARV) was adopted to support mouse ESC culture due to its promising role in allowing more complex 3D tissue formation in vitro. An increased proliferation of ESCs in HARV was observed, in keeping with their enhanced metabolic activity. Moreover, supplement of the microcarriers significantly promoted ESCs assembling into macroscopic, tissue-like organoids during the culture. The ESCs lost their Â¿stemnessÂ¿ property gradually as evidenced by their decreased expression level of undifferentiating markers over time. No significant difference of the time course of ESC differentiation was observed between the HARV and the static culture. However, an enhanced capability of their mesoderm and endoderm lineages differentiation was exhibited by the ESCs in HARV, in comparison with the static culture. We conclude that this HARV-ESC culture system might not only provide more information for the developmental biology in situ but also be directly used for tissue engineering research.