The object is to remove thrombocytes from apheresis blood. Mononuclear cells are prepared so as not to contain thrombocytes. The present invention provides a method for preparing mononuclear cells, which comprises (1) the step of mixing a non-cytotoxic and nonionic density-adjusting agent with apheresis blood to adjust density of plasma to be 1.066 to 1.078 g/ml, and (2) the step of centrifuging stratified layers including the following layers a and b to separate mononuclear cells:a. a layer of a density gradient centrifugation medium for separation of mononuclear cells,b. a layer of the density-adjusted apheresis blood layered under the layer a.
