The well-documented ability of stem cells to differentiate into various cell lineages generates tremendous potential for cell-based treatments. For example, differentiated cardiomyocytes from embryonic stem cells can be used in drug discovery processes and therapeutic cardiac treatments. Experimentation to optimize differentiation to enhance the yield of cardiomyocytes is enabled by efficient analysis of differentiation. Downstream detection technologies for such experiments vary in time, complexity, and the ability to quantitatively determine the efficiency of differentiation. Flow cytometry is a common method for detection of various cell types, but requires automated sample preparation for use in higher throughput situations. We have employed the Biomek NXP Span-8 Automation Workstation for high throughput flow cytometry sample preparation to determine the efficiency of cardiomyocyte differentiation from murine embryonic stem cells. The workflow includes fixation, permeabilization, blocking, and antibody staining in a 96-well plate format. The Agilent Microplate Centrifuge has been integrated to the system to reduce user interaction for executing the multiple washes required. The automated workflow and results from the analysis are described.