Flocculation is a desirable property in industrial yeasts and is particularly important in the fuel ethanol industry because it provides a simple and cost-free way to separate yeast cells from fermentation products. In the present study, the effect of pH and lignocellulose-derived sugars on yeast flocculation was investigated using a flocculent Saccharomyces cerevisiae, MA-R4, which has been recombinantly engineered to simultaneously co-ferment glucose and xylose to ethanol with high productivity. The flocculation level of MA-R4 dramatically decreased at pH values below 3.0 during co-fermentation of glucose and xylose. Sedimentation and microscopic observation revealed that flocculation was induced in MA-R4 when it fermented glucose, a glucose/xylose mixture, or mannose, whereas attempts to ferment xylose, galactose, and arabinose led to the loss of flocculation. MA-R4 fermented xylose and galactose more slowly than glucose and mannose. Therefore, the various flocculation behaviors shown by MA-R4 should be useful in the control of ethanol fermentation processes.