Mosaic analysis with a repressible cell marker (MARCM) generates positively labeled, wild-type or mutant mitotic clones by unequally distributing a repressor of a cell lineage marker, originally tubP-driven GAL80 repressing the GAL4/UAS system. Variations of the technique include labeling of both sister clones (twin spot MARCM), the simultaneous use of two different drivers within the same clone (dual MARCM), as well as the use of different repressible transcription systems (Q-MARCM). MARCM can be combined with any UAS-based construct, such as localized GFP fusions to visualize subcellular compartments, genes for rescue and ectopic expression, and modifiers of neural activity. A related technique, the twin spot generator, generates positively labeled clones without the use of a repressor, thus minimizing the lag time between clone induction and appearance of label. The present protocol provides a detailed description of a standard MARCM analysis of brain development that includes generation of MARCM stocks and crosses, induction of clones, brain dissection at various stages of development, immunohistochemistry, and confocal microscopy, and can be modified for similar experiments involving mitotic clones.