Recent reports describe derivation of insulincontaining cells from embryonic stem (ES) cells (1–5) and putative adult stem cells (6–8). Of particular note is the report that mouse ES cells efficiently form islet-like structures in vitro (1). Using this protocol (1) on five ES cell lines, both murine and human, we reproduced the finding that 10 to 30% of cells stain with antibodies to insulin. Fiftymicrometer clusters of insulinstaining cells were produced as described (1)(Fig. S1). Despite antibody staining, we did not detect insulin 1 mRNA by reverse transcription–polymerase chain reaction (RT-PCR) and insulin 2 mRNA detection was weak. Multiple primers used during all five stages of the protocol (1) confirmed these results. RT-PCR controls detected insulin transcripts from a single pancreatic β cell among 1 million non–β cells. Insulin gene expression was also assessed in ES cells with lacZ insertions downstream of the endogenous insulin or pdx1 promoters. Only about 1/100,000 cells was X-gal–positive despite insulin antibody staining in 10 to 30% of cells. Similarly, differentiated human ES cells expressing green fluorescent protein from an insulin promoter did not show fluorescence above background (9). Moreover, the insulin-positive cells did not stain with an antibody for C-peptide, a byproduct of de novo insulin synthesis. Nuclei of insulin-staining cells were small, condensed, and TUNEL+, suggesting apoptosis (Fig. 1, A to D). Electron microscopy of differentiated cells failed to demonstrate the granules characteristic of β cells.