Chick wing bud mesenchymal cell micromass culture allows the study of a variety of developmental mechanisms, ranging from cell adhesion to pattern formation. However, many cells remain in contact with an artificial substratum, which can influence cytoskeletal organisation and differentiation. An ultrasound standing wave trap facilitates the rapid formation of 2-D monolayer cell aggregates with a defined zero time-point, independent from contact with a surface. Aggregates formed rapidly (within 2 min) and intercellular membrane spreading occurred at points of contact. This was associated with an increase in peripheral F-actin within 10 min of cell-cell contact and aggregates had obtained physical integrity by 30 min. The chondrogenic transcription factor Sox9 could be detected in cells in the ultrasound trap within 3 h (ultrasound exposure alone was not responsible for this effect). This approach facilitates the study of the initial cell-cell interactions that occur during condensation formation and demonstrates that a combination of cell shape and cytoskeletal organisation is required for the initiation and maintenance of a differentiated phenotype, which is lost when these phenomena are influenced by contact with an artificial substrate.