Considerable evidence suggests that self-renewal and differentiation of cancer stem-like cells, a key cell population in tumorgenesis, can determine the outcome of disease. Though the development of microfluidics has enhanced the study of cellular lineage, it remains challenging to retrieve sister cells separately inside enclosed microfluidics for further analyses. In this work, we developed a photomechanical method to selectively detach and reliably retrieve target cells from enclosed microfluidic chambers. Cells cultured on carbon nanotube–polydimethylsiloxane composite surfaces can be detached using shear force induced through irradiation of a nanosecond-pulsed laser. This retrieval process has been verified to preserve cell viability, membrane proteins, and mRNA expression levels. Using the presented method, we have successfully performed 96-plex single-cell transcriptome analysis on sister cells in order to identify the genes altered during self-renewal and differentiation, demonstrating phenomenal resolution in the study of cellular lineage.