To facilitate the exploitation of embryonic stem cells (ESCs) and ESC-derived cells, scale-up of cell production and optimization of culture conditions are necessary. Conventional ESC culture methods are impractical for large-scale cell production and lack robust microenvironmental control. We developed two stirred-suspension culture systems for the propagation of undifferentiated ESCs--microcarrier and aggregate cultures--and compared them with tissue-culture flask and Petri dish controls. ESCs cultured on glass microcarriers had population doubling times (approximately 14-17 hours) comparable to tissue-culture flask controls. ESC growth could be elicited in shear-controlled stirred-suspension culture, with population doubling times ranging between 24 and 39 hours at 100 rpm impeller speed. Upon removal of leukemia inhibitory factor, the size-controlled ESC aggregates developed into embryoid bodies (EBs) capable of multilineage differentiation. A comprehensive analysis of ESC developmental potential, including flow cytometry for Oct-4, SSEA-1, and E-cadherinprotein expression, reverse transcription-polymerase chain reaction for Flk-1, HNF3-beta, MHC, and Sox-1 gene expression, and EB differentiation analysis, demonstrated that the suspension-cultured ESCs retained the developmental potential of the starting cell population. Analysis of E-cadherin-/- and E-cadherin+/- cells using both systems provided insight into the mechanisms behind the role of cell aggregation control, which is fundamental to these observations. These cell-culture tools should prove useful for both the production of ESCs and ESC-derived cells and for investigations into adhesion, survival, and differentiation phenomena during ESC propagation and differentiation.