Phosphatidylinositol-3-kinases (PI3Ks) exert a variety of signaling functions in eukaryotes. We suppressed the PI3K regulatory subunit p85a using a small interfering RNA (Pik3r1 siRNA) and examined the effects on embryoid body (EB) development in hanging drop culture. We observed a 150% increase in the volume of the treated EBs within 24 h, compared to the negative controls. Fluorescence Activated Cell Sorting (FACS) assays showed that this increase in volume is not due to increased cellular proliferation. Instead, the increase in volume appears to be due to reduced cellular aggregation and adherence. This is further shown by our observation that 40% of treated EBs form twin instead of single EBs, and that they have a significantly reduced ability to adhere to culture dishes when plated. A time course over the first 96 h reveals that the impaired adherence is transient and explained by an initial 12-hour delay in EB development. Quantitative PCR expression analysis suggests that the adhesion molecule integrin-b1 (ITGB1) is transiently downregulated by the p85a suppression. In conclusion we found that suppressing p85a leads to a delay in forming compact EBs, accompanied by a transient inability of the EBs to undergo normal cell-cell and cell-substrate adhesion.