We present for the first time a methodological approach for the measurement of cytoplasmic Zap-70 and nuclear Ki-67 proteins with QDot nanocrystal-labeled antibodies. Mononuclear cells from chronic lymphoid leukemia patients were used to detect Zap-70, and Jurkat T-cells for Ki-67. Intracellular staining kits based on saponin and paraformaldehyde were chosen as optimal for the delivery of QDot nanocrystals inside cells. Single staining with QDot 565-, 605-, or 655-labeled antibodies is operative in intracellular flow cytometry. In multicolor panels, the most robust results are obtained with the QDot 655 detected in the APC channel (633 nm excitation, 660/20 nm band pass or customized 655/20 filter) to simplify the spectral overlap compensation. In 3-, 4-, and 5-color combinations signal brightness for intracellular QDot 655 does not decrease, whereas positive-to-negative signal ratio is higher than with organic dyes. The optimized combination of fluorophores for 5-color panel includes APC-Cy7, PE, PE-Cy7, FITC, and QDot 655. We used CD3, CD19, CD38, and CD5 surface markers. Intracellular QDots outperformed organic fluorophores probed in the same samples. QDot nanocrystals are a robust tool in intracellular flow cytometry due to their narrow emission spectrum, brightness, and stability of this fluorophore.