Abstract There are two major classes of neurons in nervous systems: projection neurons and interneurons. During Drosophila nervous system development, a subpopulation of individual stem/progenitor cells gives rise to both motor neurons and interneurons. However, it remains unknown whether individual stem/progenitor cells in the mammalian brain also have the potential to give rise to both projection neurons and interneurons. Here we present evidence that single mouse neocortical progenitors generated both projection neurons and GABAergic interneurons based on studies using fluorescence-activated cell sorting (to obtain individual progenitors) and in vitro clonal analysis using time-lapse video microscopy and immunostaining. We determined that a subpopulation of individual dorsal pallial progenitors from E11.5 Dlx5/6-cre-IRES-EGFP and GAD67-GFP mice can generate both GFP-negative/Tbr1-positive (GFP−/Tbr1+)/Tuj1+ cells and GFP+/Sp8+/calretinin+/Tuj1+ cells. The GFP−/Tbr1+/Tuj1+ cells had morphological features of cultured projection neurons. Quantitative analysis of the reconstructed lineage trees derived from single progenitors showed that the projection neuron lineage appeared earlier than the interneuron lineage; however, more interneuron-like cells were produced than projection neuron-like cells. Thus, our results provide direct in vitro evidence that individual progenitors of the mammalian dorsal pallium can generate both projection neurons and interneurons.